Methods of treating allergies with M-CSF

ABSTRACT

This invention provides medical uses of a M-CSF, particularly a method and composition for treating inflammatory disease and allergy using natural M-CSF or recombinant M-CSF or the derivatives thereof.

This application is a continuation of application Ser. No. 07/730,882,filed Sep. 13, 1991, now abandoned; which was a continuation-in-part ofapplication Ser. No. 07/517,752, abandoned.

TECHNICAL FIELD

This invention relates to a method and a composition for treatinginflammatory disease and allergy.

More particularly, the present invention relates to a method of treatinga patient with inflammatory disease comprising administering an amountof macrophage-colony stimulating factor (hereinafter "M-CSF") effectiveto inhibit interleukin 1 (hereinafter "IL-1") biological activity andreduce symptoms of inflammatory disease. The present invention relatesto a method of treating a patient with inflammatory disease comprisingadministering an amount of M-CSF that is effective to cause theproduction of an inhibitor of IL-1. The present invention also relatesto a method for inhibiting IL-1 biological activity comprisingadministering a pharmaceutically effective amount of M-CSF.

This invention relates to a method of treating allergic diseasecomprising administering a pharmaceutically effective amount of M-CSF.

Further, the present invention relates to a novel anti-inflammatoryagent and anti-allergic agent, and more particularly, to ananti-inflammatory agent and anti-allergic agent containing M-CSF as anactive ingredient.

BACKGROUND ART

Inflammation is a complex of sequential changes in body tissues inresponse to injury caused by any number of agents such as bacteria,trauma, chemicals, or heat. Although the inflammatory response is amechanism of resistance by the body in the presence of such agents, theinflammatory response has caused much pain in the many patientssuffering from inflammatory diseases such as rheumatoid arthritis andgout.

The inflammatory response varies according to tissue involved, causativeagent, etc., but there are several features that characterize theresponse. These include fenestration of the microvasculature, leakage ofthe elements of blood into the interstitial spaces and migration ofleukocytes into the inflamed tissue. This is usually accompanied byclinical signs of erythema, edema, tenderness (called hyperalgesia), andpain. This complex response also involved the release and action ofchemical mediators such as histamine, 5-hydroxytryptamine (5-HT), slow,reacting substance of anaphylaxis (SRS-A), various chemotactic factors,bradykinin, and prostoglandins. In addition, phagocytic cells migrateinto the area, causing the rupture of cellular lysosomal membranes andthe release of lytic enzymes. All these events may contribute to theinflammatory response.

The mechanism of action for the inflammatory response has been the focusof interest since the earliest days of medicine. The bark of willow andother plants had been used as a medicinal agent in many cultures forcenturies. It has been reported that, in the mid-eighteenth century, theReverend Edmund Stone described the success of the bark of the willow inthe cure of "agues", i.e., fever. In 1827, Laroux discovered that theactive ingredient in willow bark is salin, which led eventually to theproduction of salicyclic acid.

The success of salicylic acid and aspirin-like drugs in alleviating manyof the symptoms of the inflammatory response is well known. These drugs,however, are not without serious side effects and disadvantages. Forexample, aspirin-like drugs inhibit or interfere with a variety of otherenzymes and cellular systems. In addition, for the treatment ofinflammatory diseases involving musculoskeletal disorders such asrheumatoid arthritis, osteoarthritis, and ankylosine soondilitis,aspirin-like drugs provide only symptomatic relief from the pain andinflammation associated with the disease and do not arrest theprogression of pathological injury to tissue. Some practitioners believethat the drugs may aggravate the disease by allowing movement ofarthritic joints that is not otherwise possible, which further promotesinjury.

Aspirin-like drugs tend to induce gastric or intestinal ulceration thatcan sometimes be accompanied by secondary anemia from the resultantblood loss.

Other drugs previously used in the treatment of inflammatory diseaseshave equally serious side effects. For example, phenylbutazone, apyrazolon derivative, was introduced in 1949 for the treatment ofrheumatoid arthritis and related disorders. Although it is an effectiveanti-inflammatory agent, it presents serious toxicity problems withlong-term use. Phenylbutazone is poorly tolerated by many patients whooften report of nausea, vomiting, epigastric discomfort and skin rashes.Diarrhea, vertigo, insomnia, euphoria, nervousness, hematuria, andblurred vision have also been reported. More serious effects includepeptic ulcers with hemorrhage or perforation, hypersensitivityreactions, ulceration stomatizis, hepatitis, nephritis, aplastic anemia,leukopenia, and agranulocytosis. In addition, a number of deaths haveoccurred, especially from aplastic anemia and agranulocytosis. Finally,because the toxic effects of the drug are more severe in elderlypersons, it is not advisable to treat the elderly with this drug.

Indomethacin, the product of a laboratory search for drugs withanti-inflammatory properties, was introduced in 1963 for the treatmentof rheumatoid arthritis and related disorders such as acute gout. It iseffective and widely used but toxicity often limits its use. It has beenreported that a very high percentage of patients (35 to 50%) experienceuntoward symptoms and about 20% must discontinue use. Gastrointestinalcomplaints include anorexia, nausea, and abdominal pain. Central nervoussystem effects include severe frontal headache, dizziness, vertigo,light-headedness, and mental confusion.

The aspirin-like drugs above are generally used, along with rest andphysical therapy, for the treatment of inflammatory diseases such asrheumatoid arthritis. If the disease progresses despite this treatment,patients are generally next treated with gold or glucocorticoids.

The toxic effects of gold, ranging from about 25 to 50% of patients withserious toxicity in about 10%, primarily involve the skin and mucousmembranes. Lesions of the mucous membranes include stomatitis,gastritis, colitis, and vaginitis. Severe blood dyscrasias may resultsfrom autotherapy. Thrombocytopenia occasionally occurs and accounts formany of the fatalities. Leukopenia, agranulocytosis, and aplastic anemiamay also occur.

Glucocorticoids, cortisol and the synthetic analogs of cortisol, arealso used in the treatment of inflammatory diseases, such as rheumatoidarthritis, because they have the ability to prevent or suppress thedevelopment of local heat, redness, swelling and tenderness by whichinflammation is recognized. Moreover, corticosteroids inhibit theinflammatory response whether the inciting agent is radiant, mechanical,chemical, infectious, or immunological. Corticosteroids, however, merelysuppress the inflammatory response and do so in such a manner that thedrugs may mask the progression of the disease. Just as importantly,corticosteroid therapy, once started, may have to be continued for manyyears or for life.

Immunosuppressive agents, developed in the search for chemotherapeuticagents for neoplastic diseases, have been explored for use ininflammatory diseases such as systemic lupus erythematosus, necrotizingvasculitis, scleroderma, and rheumatoid arthritis. One such agent,cyclophosphamide, has been the subject of a number or investigations,but it should only be used with caution. In addition to its acute toxiceffects as a nitrogen mustard, it also has a high potential for inducingsterility, teratogenic effects, mutations, and cancer.

Despite the availability of the anti-inflammatory agents above,scientists have continued to study the etiology of inflammatory diseasesin an attempt to find additional and better treatments for inflammatorydiseases. One very important recent finding is that the host response toinjury and infection involves the production of interleukin 1(hereinafter "IL-1"). IL-1 is a polypeptide immunoregulatory cytokineproduced primarily by mononuclear phagocytes that has a profound effectin the body. IL-1 mediates tissue remodeling, repair and inflammation byhelping to coordinate the activities of cells such as endothelial cells,granulocytes, osteoclasts, chondrocytes, fibroblasts, hematopoieticcells, nerve cells, and lymphoid cells. See J. W. Larrick, "Nativeinterleukin 1 inhibitors", Reviews in Immunology Today 10: 61-66 (1989).The biological activities of IL-1 include the activation of T helpercells, induction of fever, stimulation of prostoglandin or collagenaseproduction, neutrophil chemotaxis, induction of acute phase proteins andthe suppression of plasma iron levels. See U.S. Pat. No. 4,794,114 toBender, R. E. et al., "Inhibition of interleukin-1 production bymonocytes and/or macrophages", issued Dec. 27, 1988.

Recent studies have implicated excessive or unregulated IL-1 productionin exacerbating and/or causing diseases such as rheumatoid arthritis,osteoarthritis, toxic shock syndrome, Reiter's syndrome, gout, acutesynovitis, and other acute or chronic inflammatory diseases such as theinflammatory reaction induced by endotoxin. See U.S. Pat. No. 4,794,114to Bender, R. E. et al., "Inhibition of interleukin-1 production bymonocytes and/or macrophages", issued Dec. 27, 1988.

Accordingly, researchers in the field have considered treatinginflammatory disease with inhibitors of IL-1. The mechanism of IL-1inhibition varies and includes limiting IL-1 transcription, interferingwith IL-1 binding by binding directly to the IL-1 receptor, and actingat other sites to limit the activity of the IL-1 receptor.

A recent review by J. W. Larrick, entitled "Native interleukin 1inhibitors", in Immunology Today 10: 61-66 (1989) discussed IL-1inhibitors including urine-derived inhibitors (see also PCT patentapplication WO 89/01946, PCT No.PCT/US88/02819, to Dayer et al.published Mar. 9, 1989), uromodulin, monocyte-macrophage derivedinhibitors (see also Hannum, C. H., et al., "Interleukin-1 receptorantagonist activity of a human interleukin-1 inhibitor," Nature 343:336-3340 (Jan. 25, 1990); Eisenberg, S. P. et al., "Primary structureand functional expression from complementary DNA of a humaninterleukin-1 receptor antagonist," Nature 343: 341-346 (Jan. 25,1990)), and virus-infected macrophages. A more recent article hassuggested that none of these inhibitors has been purified andcharacterized. Hannum, C. H., et al., "Interleukin-1 receptor antagonistactivity of a human interleukin-1 inhibitor," Nature 343: 336-3340 (Jan.25, 1990).

Nonnative inhibitors of IL-1 have also been discussed. See, for example,U.S. Pat. No. 4,870,101 to G. Ku, entitled "Method of inhibitinginterleukin-1 release", issued Sep. 26, 1989 (providing compounds withantioxidant properties) and U.S. Pat. No. 4,780,470 to Bender et al.,entitled "Inhibition of interleukin-1 by monocytes and/or macrophages,"issued Oct. 25, 1988 (relating to the use of a4,5-diaryl-2(substituted-imidazole), U.S. Pat. No. 4,778,806, to Benderet al., entitled "Inhibition of interleukin-1 production by monocytesand/or macrophages," issued Oct. 18, 1988 (relating to the use of 2-2'1,3-propan-2-onediyl-bis(thio)!bis-1H-imadazole), U.S. Pat. No.4,794,114 to Bender et al., entitled "Inhibition of interleukin-1production by monocytes and/or macrophages," issued Dec. 27, 1988(relating to the use of a diaryl-substituted imidazole fused to athiazole pyrrolidine, thiazide or piperidine ring).

Despite the number of IL-1 inhibitors available, it is believed thatonly glucocorticoids inhibit IL-1 at transcriptional andpost-transcriptional levels. As discussed above, glucocorticoids presentserious side effects especially if implicated for long term therapy.

Thus, there is a need in the art for inhibitors of IL-1 that could beused in the treatment of inflammatory diseases. The need could be bestmet by an inhibitor of IL-1 that has been studied for its beneficialeffects in the treatment of other diseases. The side effects and anypossible toxicology as well as tolerance levels would be known for suchan inhibitor.

One of the agents of recent interest is macrophage colony-stimulatingfactor, one of the colony stimulating factors that function as ahematopoietic growth factor. This agent is promising because it has beenfound to be useful in the treatment of a variety of diseases. Forexample, M-CSF is able to promote the function of leukocytes and istherefore effective as a drug for preventing and curing variousinfectious diseases (Lopez, A. F. et al., J. Immunol., 131: 2983 (1983);Handam, E. et al., J. Immunol., 122:1134 (1979); and Vadas, M. A. etal., J. Immunol., 130: 795 (1983)). In addition, M-CSF has been found tobe effective for curing myelogenous leukemia because of itsdifferentiation inducing activity (Metcalf, D. et al., Int. J. Cancer,30: 773 (1982)). M-CSF is also thought to alleviate the leukopeniaresulting from cancer chemotherapy and radiotherapy (European PatentPublication 261592).

The use of M-CSF in the treatment of inflammatory diseases, however,could be surprising because earlier reports had suggested that thegrowth factor M-CSF would promote IL-1 production, rather than inhibitit. For example, Moore, R. N., et al. "Procedure of LymphocyteActivating Factor Interleukin 1! by Macrophages activated with colonystimulating factors, "Journal of Immunology, 125: 1302 (1980) andKurland, J. I., et al. "Induction of Prostaglandin E Synthesis in Normaland Neoplastic Macrophages; Role of Colony Stimulating Factors Distinctfrom Effects on Myeloid Progenitor Cell Proliferation, "Proceedings ofthe National Academy of Sciences 76: 2326 (1979), discuss the productionof IL-1 and prostoglandin E₂ (hereinafter "PGE₂ ") by murine peritonealmacrophages and suggest that M-CSF is implicated in the production ofthe inflammatory mediators IL-1 and PGE₂ and, thus, the progressivedevelopment of the inflammatory response.

DISCLOSURE OF THE INVENTION

The invention of the instant application overcomes the problems anddisadvantages of the art and presents an effect that is surprising inlight of the prior art by providing a method of treating a patient withinflammatory disease comprising administering an amount ofmacrophage-colony stimulating factor (hereinafter "M-CSF") effective toinhibit interleukin 1 (hereinafter "IL-1") biological activity. Thepresent invention also relates to a method of treating a patient withinflammatory disease comprising administering an amount of M-CSF that iseffective to cause the production of an inhibitor of IL-1. The presentinvention also relates to a method for inhibiting IL-1 biologicalactivity comprising administering a pharmaceutically effective amount ofM-CSF.

This invention also relates to a method of treating a patient withallergic disease comprising administering a pharmaceutically effectiveamount of M-CSF.

Based on excellent anti-inflammatory property and anti-allergic propertyof M-CSF, this invention further provides an anti-inflammatory agent andan anti-allergic agent each containing said M-CSF as an activeingredient.

Hereinafter, the above-mentioned use of treating inflammatory diseaseand the use of causing the production of an inhibitor of IL-1 will bedescribed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments ofthe invention and, together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a Northern blot analysis of macrophage mRNA that setsforth the expression of IL-1, IL-6, and TGF-β.

FIG. 2 is a radioautograph that depicts the expression of IL-1α and IL-6mRNA obtained from macrophages treated with LPS and M-CSF and acombination of LPS and M-CSF.

FIG. 3 is a graph that depicts the bioavailability of IL-1 produced bymacrophages in the presence of M-CSF, LPS, and a combination of LPS andM-CSF.

FIG. 4 is a graph that depicts the production of an IL-1 inhibitor inthe presence of LPS, M-CSF, or a combination of LPS and M-CSF.

FIG. 5 is a graph that depicts the lack of production of an IL-6inhibitor in the presence of LPS, M-CSF, or a combination of LPS andM-CSF.

FIG. 6 is a graph that depicts the separation of an IL-1 inhibitor fromIL-1 in a QAE-52 column.

FIG. 7 is a graph that depicts the ability of IL-1 and the IL-1inhibitor to bind to the IL-1 receptor.

FIG. 8 is a graph that depicts the effect of the IL-1 inhibitor on theaction of TNF.

FIG. 9 is a graph that depicts the effect of the IL-1 inhibitor and IL-1on the response of T cells to IL-2.

FIG. 10 is a graph that depicts the purification of an IL-1 inhibitor onreverse phase HPLC C-4 column and the activity of the IL-1 inhibitor interms of IL-1 bioassay, binding with the IL-1 receptor and inhibition ofbioactive IL-1.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings.

As used in the present invention, the expression "inflammatory disease"includes but is not limited to gout, rheumatoid arthritis, ankylosinespondilitis, systemic lupus erythematosus, scleroderma, Sjogren'ssyndrome, mixed connective tissue disease (MCTD), Reiter syndrome,systemic necrotizing vasculitis, hypersensitivity vasculitis, temporalarteritis, Wegener's granulomatsus, sarcoidosis, Kawasaki's disease,Buerger's disease, midline granuloma, psoriatic arthritis, inflammatorydiseases of the joints, insulin resistant diabetes, Hashimotothyroiditis, juvenile autoimmune diabetes, myasthenia gravis, ulcerativecolitis, cirrhosis and autoimmune uveitis.

Thus, the inflammatory conditions to be treated by the anti-inflammatoryagent of the invention are not limited to the above, but includeinflammatory conditions attributable to various diseases that arerepresented by the above-exemplified inflammatory diseases and includethe allergic diseases.

As used herein, the term M-CSF refers to macrophage colony stimulatingfactor. In the present invention, M-CSF that can be used in thetreatment of inflammatory diseases is one of a family of hematopoieticgrowth factors, the colony stimulating factors. Colony-stimulatingfactors (hereinafter "CSF") promote the growth of colonies ofhematopoietic cells arising from progenitor cells of bone marrow, fetalliver and other hematopoietic organs. As indicated above, the CSF ofparticular interest is macrophage colony-stimulating factor (M-CSF, alsoknown as CSF-1). M-CSF promotes the proliferation and differentiation ofthe mononuclear phagocyte lineage of cells, also known as macrophages.Unlike other CSF, such as granulocyte macrophage-CSF which is presentonly following inflammation, M-CSF is present even in noninflammatoryconditions in body fluids as a result of fibroblast production.

M-CSF refers both to naturally occurring M-CSF or recombinant M-CSF,including derivatives thereof, e.g., as described in European PatentPublications No. 261592 and No. 328061; and U.S. Pat. Nos. 4,868,119 and4,879,227. The M-CSF can be of human or any other mammalian origin, butis preferably of human origin. A particularly preferred source ofnatural M-CSF is the L929 fibroblast cells obtained from the AmericanType Culture Collection (hereinafter "ATCC").

The recombinant M-CSF and the derivatives thereof will be describedbelow in detail. The M-CSF derivatives are designated, for convenience,with reference to the amino acid primary sequence represented by theformula (1).

Formula (1): ##STR1## wherein X represents Tyr or Asp.

Thus, on the basis of the amino acid sequence from Glu at position 1 toVal at position 522 of the formula (1), there are shown variousderivatives obtained by modifying said amino acid sequence, for example,by replacements, deletions and additions. When desired, thesederivatives are abbreviated in accordance with a conventional manner.For example, M-CSF-(3-153) or M-CSF-(4-153) represents an activederivative consisting of a polypeptide having an amino acid sequencefrom Val at position 3 or from Ser at position 4 to Thr at position 153of the formula (1).

The recombinant M-CSF and the derivatives thereof are suitably used inthe invention so far as they possess the physiological activities ofM-CSF per se. Preferred are M-CSF having the total amino acid primarysequence of the formula (1), or M-CSF having an amino acid primarysequence wherein the N-terminus exists in the region from Glu atposition 1 to Glu at position 5 of the formula (1) and the C-terminusexists in the region from Thr at position 153 to Val at position 522 andwherein the N-terminus may have Met as added thereto. Particularlypreferred examples include, for example, biologically active M-CSFderivatives and recombinant M-CSF having an amino acid primary sequenceextending from Val at position 3 or Ser at position 4 to Thr at position153 or Pro at position 214 of the formula (1), which may have Met addedto the N-terminus.

In contrast to the art discussed above, as set forth in in vitro assaysto measure expression of IL-1 and IL-6 in macrophages treated with LPS,the M-CSF of the present invention does not promote the gene expressionof either IL-1 or IL-6. In addition, as measured by in vitro assays, theM-CSF of the present invention does not promote the production of IL-1,IL-6, or PGE₂ from macrophages treated with LPS.

Instead, the M-CSF of the present invention can be used in the treatmentof patients diagnosed with inflammatory disease by administration in anamount that is effective to inhibit IL-1 bioactivity. For example, asset forth in the following examples, both pretreatment and cotreatmentwith M-CSF can inhibit the production of bioactive IL-1 produced inresponse to lipopolysaccharide (hereinafter "LPS"), a bacterialendotoxin known as a potent stimulator of macrophage function in vitro.Similarly, as set forth in the following examples, pretreatment withM-CSF can inhibit the production of bioactive IL-1 produced in responseto LPS in vivo.

The M-CSF of the present invention can also be used to treat a patientdiagnosed with inflammatory disease by administering an amount of M-CSFthat is effective to cause the production of an inhibitor of IL-1. Asset forth in the following examples, administration of M-CSF appears tostimulate the production of an inhibitor of IL-1 that reduces thebioactivity of IL-1 in response to treatment with LPS in vitro. As usedherein, interleukin-1 (hereinafter "IL-1") is a factor which is producedby macrophages, particularly after interacting with immune complexes,bacterial products or T cells. IL-1 promotes thymocyte proliferation andmature T cells to release their own growth-promoting molecules. IL-1also induces fever. Fever is considered an acute response to antigenthat results in higher T cell activity.

As discussed above, IL-1 is a protein mediator of inflammation. As setforth in Larrick, J. W. et al., "The role of tumor necrosis factor andinterleukin-1 in the inflammation response," a review in PharmaceuticalResearch 5(3): 129-139 (1988), IL-1 modulates the inflammatory functionof endothelial cells, leukocytes, and fibroblasts and may promote theaccumulation of granulocytes at sites of inflammation. See also C. P. J.Maury, "Interleukin 1 and Pathogenesis of Inflammatory Diseases," ActaMed Scand 220: 291-4 (1986).

IL-1 means natural occurring IL-1α, naturally occurring IL-1β,recombinant IL-1α, recombinant IL-1β, and derivatives of IL-1α or IL-1βsuch as described in European Patent Publication 237073, European PatentPublication 237967, PCT Publication 89/01946 (also see Oppenheim, J. J.et al., Immunol. Today 7: 45 (1986)). The IL-1 can be of human or anyother mammalian origin, preferably human origin.

As used herein, the term "IL-1 inhibitor" refers to an agent that isable to inhibit the biological activity of IL-1, such as in themediation or progression of inflammation. Inhibition of biologicalactivity can be assessed using any number of assays for IL-1 activitywith which those of ordinary skill in the art would be familiar, such asthe IL-1 bioassay described by Togawa, A. et al., "Characterization ofLymphocyte Activating Factor Produced by Human Mononuclear Cells;Biochemical Relationship of High and Low Molecular weight Forms of LAF,"J. Immunol. 122: 2112 (1979).

Additionally, the IL-1 inhibitor of the invention refers to an agentthat is able to bind to the IL-1 receptor. Such a characteristic can beeasily determined by those of ordinary skill in the art, usingtechniques, for example, as described by Kilian, T. L., et al.,"Interleukin 1 Alpha and Interleukin 1 Beta Bind to the Same Receptor onT Cells," J. Immunol. 136: 4509 (1986).

The production of the IL-1 inhibitor of the present invention isaffected by M-CSF. In a preferred embodiment, the IL-1 inhibitor isregulated by M-CSF so that administration of increasing doses of M-CSFcauses a greater inhibition in IL-1 bioactivity.

In an additional preferred embodiment, the inhibitor is specific to IL-1and will not regulate the activity of IL-2, IL-6 or TNF (tumor necrosisfactor).

The present invention relates to the inhibition of IL-1 biologicalactivity by administering a pharmaceutically effective amount of M-CSF.As used herein, pharmaceutically effective refers to an amount of anagent that is able to reduce any of the symptoms of the inflammatorydiseases described above. As is known by those of ordinary skill in thisart, symptoms of inflammatory disease include erythema, edema,tenderness (hyperalgesia), and pain. The dosage of M-CSF that may reducethese symptoms range from 1 μg/kg to 100 mg/kg, with a particularlypreferred dosage of 1 mg/kg to 35 mg/kg.

Persons of ordinary skill in the art would be able to optimize thedosage of the M-CSF or IL-1 inhibitor of the instant invention usingtechniques that are known in the art. Those techniques are set out, forexample, on pages 19-28 of Goodman and Gilman, The Pharmacological Basisof Therapeutics, 5th Ed. (1975). Dosages can be ascertained andoptimized through the use of the established assays and conventionaldose-response studies. Further refinements of the calculations necessaryto determine the appropriate dosage for treatment are routinely made bythose of ordinary skill in the art and are within the array of tasksroutinely performed by them without undue experimentation. In addition,the dosage of M-CSF or IL-1 inhibitor to be administered in the methodsof the present invention will vary depending upon, for example, theparticular inflammatory disease to be treated, the mode ofadministration, the age, the weight and the sex of the subject to beadministered.

The present invention envisions any mode of administering either M-CSFor the IL-1 inhibitor. Examples of such modes of-administration includeintravenous, intramuscular, local, transdermal, injection, oral andparenteral. A preferred administration is a local administration to thesite.

The M-CSF and IL-1 inhibitor of the present invention can beadministered alone or in combination with any of the conventionalanti-inflammatory agents discussed above.

Appropriate pharmaceutically acceptable carriers, diluents and adjuvantscan be used together with the compounds described herein to prepare thedesired compositions for use in the treatment of patients. Thepharmaceutical compositions of this invention will contain the M-CSF orthe IL-1 inhibitor compound together with a solid or liquidpharmaceutically acceptable nontoxic carrier. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. In addition, the compounds of the instantapplication can be associated with liposomes, as set forth in copendingU.S. application Ser. No. 07/505,584, to Gideon Strassmann, filed Apr.6, 1990, now abandoned hereby specifically incorporated by reference.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesiumcarbonate, magnesium stearate, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene glycol,water, ethanol and the like. These compositions can take the form ofsolutions, suspensions, tablets, pills, capsules, powders,sustained-release formulations and the like. Suitable pharmaceuticalcarriers are described in "Remington's Pharmaceutical Sciences" by E. W.Martin. Such compositions will contain an effective therapeutic amountof the active compound together with a suitable-amount of carrier so asto provide the form for proper administration to the patient. Thecompounds of the present invention can be used in the treatment ofpatients. Patients shall be used in its broadest sense to mean mammals,including humans, as well as animals, for example, dogs, cats, guineapigs, mice, and rats.

As already stated, this invention also relates to a use of ananti-allergic composition comprising, as an active ingredient, the M-CSFspecified in this specification including M-CSF derivatives such asbiologically active recombinant human M-CSF derivatives having an aminoacid sequence extending from Val at 3-position or Ser at 4-position toThr at 153-position or Pro at 214-position of the formula (1), which mayhave Met added to the N-terminus.

Hereinafter, the process for preparing a M-CSF-derivative used as anactive ingredient in the invention are described in detail. Saidderivative is prepared utilizing a gene coding for the same, i.e. bypreparing a recombinant DNA for the expression of said gene in hostcells, introducing the DNA into host cells for transformation thereofand cultivating the resulting transformant.

The gene for the production of a human M-CSF derivative of thepresent-invention can be prepared starting with mRNA isolated fromvarious human cells having ability to produce M-CSF, more specificallyand advantageously from the AGR-ON human leukemic T cell-derivedcultured cell line having the characteristics described in UnexaminedJapanese Patent Publication SHO 59-169489; deposited in the AmericanType Culture Collection (ATCC) under the ATCC deposition No.CRL-8199!.The extraction procedure for the isolation of mRNA from said AGR-ON canbe carried out, for example by the guanidinium/hot phenol method T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning, pp. 194-195(Cold Spring Harbor Laboratory), 1982!, the guanidinium/cesium chloridemethod T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning, p.196 (Cold Spring Harbor Laboratory), 1982! or the like. The conversionof the purified mRNA to cDNA, namely the synthesis of the desired gene,can be realized, for example by the Okayama-Berg method H. Okayama andP. Berg, Molecular and Cellular Biology, vol. 3, p. 280 (1983)!, theGubler-Hoffman method V. Gubler and B. J. Hoffman, Gene, vol. 25, pp.263-269 (1983)! or the like. The transformation by introducing thethus-obtained DNA into host cells and the selection of a desired straincarrying the M-CSF cDNA from among the transformants can be performed byconventional methods. Unexamined Japanese Patent Publication HEI1-104176 describes these procedures or methods in detail. The M-CSF genedescribed in said patent specification also can be advantageouslyemployed as the M-CSF gene to be used in the practice of the presentinvention.

The genes mentioned above can also be prepared in the manner of chemicalsynthesis of nucleotides using a conventional method such as thephosphite triester method Nature, 31, 105 (1984)!. In any of themethods, various procedures or steps, such as partial DNA synthesis bychemical means, enzyme treatments for DNA chain cleavage, deletion,addition and ligation, DNA isolation, purification and replication,screening, etc., can be performed in the conventional manner. Forexample, the above-mentioned DNA isolation and purification can becarried out by agarose gel electrophoresis, etc., and partialmodification of a codon in the nucleic acid sequence can be conducted bysite-specific mutagenesis Proc. Natl. Acad. Sci., 81, 5662-5666 (1984)),etc. The codon to be selected for a desired amino acid is not limitedspecifically but can be determined in a usual manner in view of thecodon usage for the host cell to be utilized, etc. The DNA sequence ofthe desired gene to be used in the above methods can be determined andconfirmed, for example, by the Maxam-Gilbert chemical modificationmethod Methods in Enzymolozy, 65, 499-560 (1980)! or by thedideoxynucleotide chain termination method using M13 phage Messing, J.and Vieira, J., Gene, 19, 269-276 (1982)!.

The M-CSF derivatives of the present invention can be produced easily inlarge Quantities by recombinant DNA techniques using the genes obtainedin the above manner. While the use of the above-mentioned specific genesis essential, this process can be conducted basically by general geneticengineering techniques cf. Molecular Cloning, T. Maniatis et al., ColdSpring Harbor Laboratory (1982), etc.!.

More specifically, a recombinant DNA which enables the expression of anM-CSF gene in host cells is produced, and the DNA is introduced into thehost cell for transformation thereof and the transformant is cultivated.

Useful host cells can be either eukaryotic or prokaryotic cells.Generally used as the prokaryotic hosts are E. coli and Bacillussubtilis. In the practice of the invention, a plasmid vector capable ofreplication in such a host, for instance, is employed and, for enablingexpression of an M-CSF gene in said plasmid vector, an expression vectorcan be used which contains a promoter and an SD (Shine-Dalgarno)sequence and further an initiation codon (e.g. ATG) required for theinitiation of protein synthesis upstream from said gene. Widely used ashost E. coli is the strain K12. pBR322 is generally used as the vector.However, these are not limitative but various known strains and vectorscan be used. Useful as the promoter are, for example, the tryptophan(trp) promoter, lpp promoter, lac promoter, P_(L) promoter, etc. and, inany case, the desired gene can be expressed.

As a preferred process for producing the human M-CSF derivatives of thepresent invention using the genes mentioned above, there may bementioned, by way or example, a process which uses a prokaryotic host,such as E. coli, as the host and in which the desired protein isexpressed by the two-cistron technique. This is a gene expression systemcomprising two cistrons in sequence and the process yields andaccumulates the desired human M-CSF derivative stably in largequantities in host cells.

The production of an M-CSF derivative of the present invention by saidtwo-cistron method is now described in detail. First, an expressionplasmid is prepared which contains two cistrons, namely a gene as thefirst cistron coding for an appropriate polypeptide and an M-CSF gene asthe second cistron. It is important that the plasmid should contain,upstream from the first cistron, a promoter and an SD sequence for theexpression of said gene and further contain, downstream from the firstcistron but upstream from the second cistron, a synthetic linker whichcontains an SD sequence for the expression of the second cistron, atermination codon for the first cistron and an initiation codon for thesecond cistron, as arranged in this order.

The gene to be used as the first cistron may be a synthetic or naturalgene insofar as the gene can be expressed in the host. Since theexpression of said gene leads to production of a polypeptide differentfrom the M-CSF derivative in the same system and this makes it necessaryto separate said polypeptide, it is desirable that said polypeptideshould be hydrophobic and have a molecular weight greatly different fromthat of the M-CSF derivative. It is desired that the above-mentionedfirst cistron should code for such a polypeptide. As preferred examplesof the first cistron, there may be mentioned genes coding for IL-2,IFN-α, -β and -γ, etc. and fragments derived from these genes and codingfor about 50 to 100 amino acid residues.

The promoter and SD sequence to be arranged upstream from the firstcistron may be per se known ones. Examples of such promoter are the trppromoter, tac promoter, P_(L) promoter, P_(R) promoter, lpp promoter,OmpA promoter, lac promoter, etc., among which the trp promoter, P_(L)promoter, P_(R) promoter and the like are particularly preferred.Examples of the SD sequence are sequences of 3 to 9 base pairs, such asGGAG and AGGA, that are capable of forming a hydrogen bond with the 3'terminus of 16S rRNA of prokaryotic cells.

The initiation codon and the termination codon to be present in thesynthetic linker between the first cistron and the second cistron can beany of the naturally occurring ones. These codons need not be used eachin a complete form, for example as TGA and ATG, but can be used in apartly overlapping form, for example as TGATG, TAATG, etc. Theexpression plasmid for use in the above two-cistron method can beconstructed by a usual method.

A method, which can be mentioned as a particularly preferred example,comprises first cleaving an M-CSF gene-containing plasmid with asuitable restriction enzyme, isolating and purifying the resulting M-CSFgene-containing fragment by a usual method, separately synthesizing theabove-mentioned synthetic linker by a usual method, for example by usinga DNA synthesizer, joining the linker to the fragment obtained in theabove manner on the upstream side of the M-CSF gene using T4 DNA ligaseor the like, and incorporating the resulting DNA fragment into a plasmidcontaining the first cistron and capable of expressing the same, at aproper position. Alternatively, an expression plasmid suitablefor-the-desired two-cistron system can be prepared from the DNA fragmentobtained by the above method and containing the second cistron as joinedto the synthetic linker, by joining the first cistron to the upstreamend of said fragment, and introducing the resulting DNA fragment into aplasmid having a suitable protein expression system.

The plasmid thus obtained is introduced into suitable host cells fortransformation of the cells, whereby the desired transformant can beobtained through the two-cistron method. When such transformant is used,the protein relating to the first cistron and the desired M-CSFderivative relating to the second cistron can be expressed individually.These products can be analysed or identified by a usual method, forexample by SDS-PAGE, Western blotting or the like, and can be separatedfrom each other and purified by the various methods described laterherein.

The desired transformant thus obtained can be cultivated by a usualmethod, whereby the human M-CSF derivative is produced and accumulated.The medium to be used for said cultivation may be one suitably selectedfrom among those usually used for the host cell employed. Forcultivating the transformant using E. coli or the like as the host, forexample, LB medium, E medium, M9 medium, M63 medium and the like can beused. Various carbon sources, nitrogen sources, inorganic salts,vitamins, cell extracts, physiologically active substances, etc., whichare generally known, can be added to these media when required.

In cultivating the above transformant, those cultivation conditions thatare suited for the growth of host cells can be employed. In the case ofE. coli, for instance, a pH of about 5-8, preferably 7 or about 7, and atemperature of about 20°-43° C., preferably 37° C. or about 37° C., canbe employed.

In the above manner, the desired M-CSF derivative of the presentinvention is produced and accumulated in the transformant cells. Thisderivative can be isolated and purified by various separation proceduresutilizing the physical, chemical or other properties thereof. As typicalexamples of said procedures, there may be mentioned usual refoldingtreatment, treatment with a protein precipitating agent, centrifugation,osmotic shock method, ultrasonication, ultrafiltration, molecular sievechromatography (gel filtration), adsorption chromatography, ion-exchangechromatography, affinity chromtography, high-performance liquidchromatography (HPLC), various other liquid chromatographic techniques,dialysis, and combinations of these. By using such separationprocedures, the desired human M-CSF derivative can be produced easily inhigh yields with high purity on a commercial scale.

The thus-obtained M-CSF derivatives of the present invention are incommon in that they have CSF activity although they differ slightly inN-terminal amino acid sequence, molecular Freight, etc. depending on thegene used for the production and on the expression system for theexpression of said gene and, especially, may have a Met residuecorresponding to the initiation codon as added to the N-terminus in theprocess of expression of the derivatives of the invention.

Preferred as the active ingredient of the anti-allergic compositionaccording to the invention are M-CSF derivatives wherein the N-terminusexists in any position of the region from Glu at 1-position to Glu at5-position of the formula (1) (said N-terminus may have Met as addedthereto), and wherein the C-terminus exists in any position of theregion from Thr at 153-position to Val at 522-position of the formula(1).

Of these derivatives, particularly preferred are, as stated above, M-CSFderivatives having an amino acid primary sequence extending from Val at3-position or Ser at 4-position to Thr at 153-position or Pro at214-position of the formula (1), wherein the N-terminus may have Met asadded thereto.

Also usable is a M-CSF derivative having a total amino acid sequenceextending from Met at position -32 to Val at position 522 of the formula(1).

The anti-allergic agent of the invention is usually formulated into apharmaceutical composition containing an effective amount of M-CSFincluding the M-CSF derivatives obtained above in combination with aconventional pharmacologically acceptable non-toxic carrier, and isadministered by various routes depending on the form of the composition.

Such compositions are for example, in the form of liquid preparationsincluding solutions, suspensions, emulsions and the like, which areusually given orally, intravenously, subcutaneously, intracutaneously orintramuscularly. Such forms or methods of administration are not limitedspecifically. The derivatives can also be prepared in other variouspreparation forms usually used and suited, for example, to oral orparenteral administration. The compositions can be provided also as drypreparations which can be reconstituted to liquids by addition of asuitable carrier. While the dose of each preparation is not limitedspecifically but can be determined suitably depending on the desiredpharmacological effect, the kind of disease, the age and sex of thepatient, the severity of disease, etc., the preparation is administeredgenerally at such a dose that the active ingredient is administered inan amount of about 0.001 to about 1 mg/kg/day calculated as the amountof protein. The composition may be given daily either in a single doseor in several divided doses.

When a biologically active M-CSF derivative having an amino acid primarysequence extending from Val at 3-position or Ser at 4-position to Thr at153-position of the formula (1) which may have Met as added to theN-terminus is used as the active ingredient M-CSF in the invention, theresulting composition not only possesses its own anti-inflammatory andanti-allergic properties, but also has a feature of exhibiting a higherbioavailability compared with other M-CSF. Thus such anti-inflammatoryand anti-allergic compositions are very useful from this view point aswell.

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention.

In the following EXAMPLES and REFERENCE EXAMPLES, each of the M-CSFderivatives designated with reference to the amino acid sequence of theformula (1) is one wherein X in said formula represents Asp.

EXAMPLE 1 M-CSF does not stimulate the production of the inflammatorymediators IL-1, IL-6, and PGE₂

As set forth in FIGS. 1 and 2 and Table 1, M-CSF does not promote theproduction in macrophages of the inflammatory mediators IL-1α, PGE₂, andIL-6.

A. M-CSF does not promote expression of IL-1 or IL-6

1. The experimental model

Lipopolysaccharide (hereinafter "LPS"), the active component ofendotoxin derived from bacterial cells, was employed as the model systemto demonstrate the effects of M-CSF on the production of inflammationmediators in inflammatory macrophages.

To obtain inflammatory macrophages, macrophages were harvested frominbred female pathogen-free C57B1/6 mice (Charles River BreedingLaboratories, Wilmington, Mass.) 3 days after the mice were injectedwith 1.0 ml of sterile Brewer's thioglycollate (TG). The cells wereobtained by peritoneal lavage with 10 ml of ice cold HBSS (Hanks'Balanced Salt Solution) supplemented with 10 U/ml of heparin andcentrifuged at 250×g for 5 min. at 4° C.

The resulting cell pellet was resuspended in RPMI 1640 medium (GIBCO,Grand Island, N.Y.) supplemented with 10% (v/v) of heat-inactivated, lowendotoxin (0.008 ng/ml), fetal calf serum (Hyclone, Logan, Vt.), 2.0 mMglutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin(hereinafter "complete medium").

1.0 ml aliquots of 0.5×10⁶ macrophages were plated per 16 mm well(2.5×10⁵ macrophages/cm²) in tissue culture dishes (Costar, Cambridge,Mass.). The dishes were incubated for 2.0 hours at 37° C. in 6.0% CO₂,and washed 3 times with warm (37° C.) complete medium to removenon-adherent cells. The cell monolayers contained approximately 95%macrophages, as determined by non-specific esterase staining.

The cells were exposed at 37° C. to LPS and M-CSF as set forth below insections 2 and 3. The resulting conditioned medium was collected bycentrifugation at 570×g for 15 min at 4° C., dialyzed against 100volumes of RPMI 1640 medium, filter sterilized and stored at -35° C. forfurther analysis.

2. M-CSF does not promote expression of IL-1 or IL-6

The inflammatory macrophages were exposed to a control; to LPS (0.5μg/ml obtained from E. coli 0.55:B5 (Difco, Detroit, Mich.)); and toM-CSF (0.5 μg/ml of recombinant M-CSF-(3-153) with specific activity of2.7×10⁷ U/mg (Batch 058-2) produced as described in Takahashi, M. etal., Biochemical and Biophysical Research Communications 161 (2):892-901 (Jun. 15, 1989) and purified as in Reference Example 4 to bedescribed below for varying times as follows. 1.0 unit of M-CSF isdefined as the half maximal stimulation-of growth of the M-CSF dependenthuman cell line NFS-60, Nakoinz, I. et al., Exp. Hemotol. 17: 669(1989).

    ______________________________________                                        Tube      Contents        Time (hours)                                        ______________________________________                                        1         Control         6.0                                                 2         LPS (5 μg/ml)                                                                              0.75                                                3         LPS (5 μg/ml)                                                                              1.5                                                 4         LPS (5 μg/ml)                                                                              3.0                                                 5         LPS (5 μg/ml)                                                                              6.0                                                 6         M-CSF (058-2 0.5 μg/ml)                                                                    0.75                                                7         M-CSF (058-2 0.5 μg/ml)                                                                    1.5                                                 8         M-CSF (058-2 0.5 μg/ml)                                                                    3.0                                                 9         M-CSF (058-2 0.5 μg/ml)                                                                    6.0                                                 ______________________________________                                    

IL-6 and TGF-β using the Northern Analysis of macrophage mRNA. Such ananalysis is among the routine skills of those in this art and isdescribed, for example in Section XI., Nucleic Acid Hybridization:Determination of Genetic Homology in Recombinant DNA Methodology, DillonJ. R., A. Nasim, E. R. Nestmann (eds) John Wiley & Sons (1985).

The sections designated IL-1α and IL-6 of FIG. 1 indicate that LPS canpromote the expression of both IL-1α and IL-6. Compare the control(lane 1) with samples that received LPS alone (lanes 2-5). Theexpression of both IL-1α and IL-6 increased with time but peaked at 3hours.

In contrast, the presence of M-CSF (lanes 7-10) did not promote theexpression of either IL-1α or IL-6. Both those lanes are blank.

As can be seen in the section designated TGF-β, TGF-β was expressed byLPS, as well as M-CSF, indicating that all the proteins were active.

This experiment was repeated twice with identical results. Thus, unlikeLPS which does promote the production of inflammatory mediators, M-CSFdoes not stimulate the production of IL-1 or IL-6. This finding issurprising in light of earlier reports that indicated that M-CSFstimulated the production of IL-1.

3. M-CSF does not stimulate IL-1α gene expression

An experiment conducted with a different preparation of M-CSF confirmedthe inability of M-CSF to induce gene expression of IL-1. In FIG. 2, adifferent preparation of M-CSF was tested and found not to stimulateIL-1α expression.

Inflammatory macrophages were exposed to a control (lane 1), to LPS at 1μg/ml (obtained as above) (lane 2), to M-CSF-(3-153) at 1 μg/ml(obtained as above except a different batch with a specific activity of3.0×10⁷ U/mg) (lane 3), and to a combination of LPS and M-CSF (lane 4).The cells were analyzed for expression of IL-1α as set forth inMolecular cloning: a laboratory manual, T. Maniatis, E. F. Fritsch, J.Sambrook (eds) Cold Spring Harbor Press (1982).

Compared to the control lane (1), LPS (in lane 2) did stimulate theexpression of IL-1α gene, M-CSF (in lane 3) did not stimulate theexpression of IL-1α, and the addition of M-CSF to LPS (in lane 4) didnot affect the expression of IL-1α. Thus, as set forth in section 1above, M-CSF neither induces nor reduces the expression of IL-1α.

B. M-CSF does not promote the activity of IL-1, IL-6, or PGE₂

1. The experimental model

a. Conditioned media

Inflammatory macrophages, obtained as set forth above, were treated withM-CSF from two sources to yield conditioned media as follows:

recombinant M-CSF-(3-153) (Batch EC-80-I) with specific activity of3.0×10⁷ U/mg and produced as above; and

natural M-CSF with a specific activity of 1.5×10¹⁰ U/mg derived fromL929 fibroblasts (obtained from E. R. Stanley (Albert Einstein Collegeof Medicine, NY)).

The cells were exposed to increasing doses of both sources of M-CSF upto a maximum dose of 1 μg/ml. After an incubation period of 24 hours,the conditioned medium was analyzed for the activity of IL-1, IL-6, andPGE₂ as set forth below.

b. The IL-1 thymocyte assay

To assess the effect of the conditioned media on the production ofbioactive IL-1, the cells were subjected to a thymocyte assay asdescribed by Togawa, A., et al., "Characterization of LymphocyteActivating Factor Produced by Human Mononuclear Cells; BiochemicalRelationship of High and Low Molecular Weight Forms of LAF," J. Immunol.122: 2112 (1979). In this assay, a single cell suspension of thymocytes,obtained from female C3H/He mice (Bantin-Kingman (CA)) 6 to 10 weeks ofage, was prepared by gentle mincing. Large aggregates were removed byincubation on ice for 10 min. at unit gravity. The cells were washedtwice with complete medium and resuspended to a density of 1.0×10²cells/ml in complete medium. The cells were thereafter seeded inflat-bottomed microtiter plates at a density of 1.0×10⁶ cells/well inthe absence or presence of 1 μg/ml of Concanavalin A (hereinafter"ConA") (Sigma Chemical Co.).

Either conditioned medium or recombinant human IL-1β was added in serialdilutions to a final volume of 0.2 ml. The thymocytes were cultured for72 hours and pulsed with 1.0 μCi of methyl!-³ H-TdR (6.7 Ci/Mmol, NewEngland Nuclear, Boston, Mass.). ³ H-TdR incorporation was determined bystandard liquid scintillation counting procedures after harvest of thecells on glass fiber paper using a semiautomatic cell harvester(Skatron, Sterling, Va.).

As set forth in Table 1 below, neither the natural M-CSF (designatedM-CSF Type L929) nor the recombinant M-CSF-(3-153) (designated E. coli3-153!M-CSF or rh-M-CSF (3-153)) exhibited positive results in theassay, indicating the neither source of M-CSF could produce IL-1 ininflammatory macrophages.

c. The IL-1 radioreceptor assay

The observation that both human IL-1α and IL-16 bind to the samereceptor as murine IL-1 on murine EL-4.6.1 target cells led to thedevelopment of the IL-1 receptor assay described by Kilian, T. L., etal., "Interleukin 1 Alpha and Interleukin 1 Beta Bind to the SameReceptor on T Cells," J. Immunol. 136: 4509 (1986). In the assay, murineEL-4.6.1 cells from the ATCC, maintained in complete medium, werecentrifuged and resuspended in binding buffer comprising RPMI 1640medium supplemented with 0.1 mg/ml of bovine serum albumin and 25 nMHEPES (pH 7.2). Cell density was adjusted to 8.0×10⁶ cells/ml and 0.05ml aliquots were dispensed into tubes placed on ice.

275 pg of ¹²⁵ I-IL-1α (75000 cpm) (specific activity, 2000 Ci/mmol;Amersham, Arlington Heights, Ill.) was added to each tube. The tubeswere then incubated for 3.0 hours at 4° C. with diluted conditionedmedium from macrophages treated with up to 1.0 μg/ml of the two sourcesof M-CSF.

Non-specific binding was determined by the addition of 5.8 pMrecombinant human IL-1β (specific activity, 4.0×10⁷ U/mg; obtained fromHirai (Otsuka Pharmaceutical, Tokushima, Japan)). To separate ¹²⁵I-IL-1α which was bound to the cells from free ¹²⁵ I-IL-1α, 0.2 ml of anoil mixture of dibutyl phthalate and dioctyl phthalate (10:1 (v.v)) wasinjected into each assay tube and each tube was centrifuged for 2.0 minat 4° C. in a macrofuge. The fluid was then aspirated, the tube tipswere cut and cell bound radioactivity was determined in a gamma counter.

As set forth in Table 1 below, neither type of M-CSF could displace theIL-1, indicating that neither type of M-CSF stimulated the production ofIL-1 in the macrophages.

d. IL-1 mRNA assay

The expression of IL-1 mRNA was determined as above. As in FIG. 1, Table1 indicates that neither the recombinant M-CSF nor the natural M-CSF wasable to promote the expression of IL-1 from the macrophages, confirmingthat neither type stimulated the production of IL-1 in the macrophages.

e. IL-6 B-9 assay

The IL-6 assay employed was that described by Aarden et al., Eur. J.Immunol., 17: 1411-1416 (1987) as follows. The IL-6 dependent hybridomacell line B13.29 clone B9 was maintained in Iscove's modified Dulbecco'smedium supplemented with 2.0% (v/v) MG63 osteocosarcoma from ATCCconditioned medium as a source of IL-6. To these cells was added 5.0%(v/v) fetal calf serum, 50 μM β-mercaptoethanol, 100 U/ml of penicillinand 100 mg/ml of streptomycin. 5.0×10⁴ /ml of clone B9 cells werecultured in 0.2 ml of above medium in flat-bottomed 96 well plates(Falcon). Conditioned media was added to the cells and the plates wereincubated for 72 hours in an atmosphere of 5.0% CO₂, in air at 37° C.The plates were incubated with 1.0 μCi ³ H!-thymidine for the last 4hours of the culture period. The cells were then harvested using a cellharvester (Skatron, Sterling, Va.) and the ³ H!-thymidine which had beenincorporated into DNA was quantitated by a liquid scintillation counter(LKB RockBeta). The samples were tested in duplicate, titrated in 3-folddilutions. All of the assays were compared to a standard curve using1.0×10⁷ U/mg of recombinant human IL-6 (Amgen, Thousand Oaks, Calif.).

As set forth in Table 1, neither the recombinant M-CSF nor the naturalM-CSF was able to stimulate the production of IL-6, indicating theneither form could stimulate the production of IL-1 in macrophages.

f. IL-6 mRNA assay

The expression of IL-6 mRNA was determined as above and confirmed theresults obtained with the B-9 assay. As set forth in Table 1, neitherform of M-CSF was able to stimulate the expression of IL-6.

g. PGE₂ assay

In order to determine the ability of M-CSF to stimulate the productionof PGE₂, the presence of PGE₂ was determined as follows. The conditionedmedium from macrophages treated with up to 1.0 μg/ml of both recombinantand natural M-CSF was diluted with phosphate buffered saline (PBS),acidified to approximately pH 2 and extracted with an equal amount ofchloroform:methanol (1:1 (v/v)). The organic phase was dried undernitrogen and quantification of PGE₂ was determined by a standardizedradioimmunoassay kit purchased from New England Nuclear, Boston, Mass.

As set forth in Table 1 below, as with IL-1 and IL-6, neither form ofM-CSF could stimulate the production of PGE₂.

                  TABLE 1                                                         ______________________________________                                        Production of Inflammatory Mediators by Macrophage in                         Response to M-CSF                                                             Inflammatory         M-CSF Type                                               Mediators                                                                              Assay       L929       rh M-CSF (3-153)                              ______________________________________                                        IL-1α                                                                            Thymocyte   No         No                                                     Radioreceptor                                                                             No         No                                                     mRNA        No         No                                            PGE.sub.2                                                                              RLA         No         No                                            IL-6     B-9         No         No                                                     mRNA        No         No                                            ______________________________________                                    

As indicated by the above experiments, M-CSF does not stimulate theproduction of inflammatory mediators such as IL-1, IL-6, or PGE₂.

EXAMPLE 2

M-CSF regulates the production of inflammatory mediators produced inresponse to LPS treatment As set forth in FIGS. 2-10 and Tables 2-3 andas set forth below in detail, M-CSF is able to decrease the activity ofthe inflammatory mediators IL-1 and PGE₂ produced in response to LPS.

A. M-CSF regulates the production of PGE₂

1. Pretreatment with M-CSF reduces PGE₂ production

Macrophages were obtained as in Example 1 and divided into four sets.Two sets of macrophages were pretreated with culture medium and two werepretreated with recombinant M-CSF obtained as in Example 1. In thetreatment phase, one set of cells pretreated with medium and one set ofcells pretreated with M-CSF received LPS at 1 μg/ml as set forth inTable 2 below. The other set received medium. The presence of PGE₂ wasdetermined as described in Example 1, and expressed in ng/mg(cellprotein)/24 hr. or ng/ml/24 hr.

As set forth in Table 2a, the control levels of PGE₂ when pretreatedwith either medium or M-CSF is 0.11-0.12. LPS treatment of cellspretreated with medium yields PGE₂ levels of approximately 10.00, anearly 100 fold increase over control levels. When LPS is added to cellspretreated with M-CSF, however, the increase in PGE₂ levels is inhibitedby 65% to only a level of approximately 3.6.

Thus, it appears that pretreatment with M-CSF Will block the capacity ofLPS to stimulate PGE₂ levels.

                  TABLE 2a                                                        ______________________________________                                        Pretreatment or Co-Treatment of M-CSF Reduces LPS induced                     PGE.sub.2 Formation by Macrophages                                                                PGE.sub.2                                                 Pretreatment                                                                           Treatment  (ng/mg/24 h.) ± S.D.                                                                      % Inhibition                               ______________________________________                                        Medium   Medium     0.11           --                                         Medium   LPS (1 μg/ml)                                                                         10.0           --                                         M-CSF (rh)**                                                                           Medium     0.12           --                                         M-CSF (rh)**                                                                           LPS (1 μg/ml)                                                                         3.6            65                                         ______________________________________                                         ** E. coli 3153!MCSF (MCSF-(3-153))                                           2. Pretreatment or cotreatment with MCSF reduces PGE.sub.2 production    

Macrophages obtained as in Example 1 were pretreated either with culturemedium or M-CSF as set forth in Table 2 below. Both recombinantM-CSF-(3-153) (designated "rh") obtained as set forth above in Example 1and natural M-CSF (designated "L929") obtained as set forth above inExample 1, were used to pretreat the cells.

The cells were then treated Keith medium (to obtain control levels ofPGE₂), both sources of M-CSF (to demonstrate the effect of treatmentwith different M-CSF samples), LPS alone at a concentration of 1 μg/ml,and a combination of LPS with both sources of M-CSF, as set forth belowin Table 2. The cells were analyzed for presence of PGE₂ produced inng/ml/24 hr. This experiment was repeated five times.

As shown in Table 2, the level of PGE₂ produced was 0.07 in the controlcells, was 0.19 and 0.07 in the cells that were only pretreated withM-CSF, and was 0.06 and 0.1 in the cells that were only treated withM-CSF. In the cells pretreated only with medium, treatment with LPSincreased the levels to 1.27 and 1.10. In contrast, in cells that weretreated with LPS and either pretreated or cotreated with M-CSF,production of PGE₂ was inhibited by 48%, 87-89%, or 100%. Thus, eitherpretreatment or cotreatment with M-CSF will decrease the production ofPGE₂ stimulated by LPS.

                  TABLE 2                                                         ______________________________________                                        Pretreatment or Co-Treatment of M-CSF Reduces LPS induced                     PGE.sub.2 Formation by Macrophages                                            Pre-                  PGE.sub.2                                               treatment Treatment   (ng/ml/24 h) ± S.D.                                                                     % Inhibition                               ______________________________________                                        Medium    Medium       0.07 ± 0.0035                                       Medium    LPS (1 μg/ml)                                                                          1.27 ± 0.09                                          Medium    M-CSF (L929)*                                                                             0.06 ± 0.005                                                                            --                                         Medium    M-CSF (rh)**                                                                               0.1 ± 0.009                                                                            --                                         Medium    LPS +       0.7 ± 0.03                                                                              48                                                   M-CSF (L929)*                                                       Medium    LPS +        0.2 ± 0.001                                                                            89                                                   M-CSF (rh)**                                                        Medium    Medium      0.07 ± 0.003                                                                            --                                         M-CSF (rh)**                                                                            Medium      0.190 ± 0.009                                                                           --                                         M-CSF (L929)*                                                                           Medium      0.07 ± 0.004                                                                            --                                         Medium    LPS (1 μg/ml)                                                                          1.10 ± 0.02                                                                             --                                         M-CSF (rh)**                                                                            LPS (1 μg/ml)                                                                           0.2 ± 0.001                                                                            87                                         M-CSF (L929)*                                                                           LPS (1 μg/ml)                                                                          0.07 ± 0.004                                                                            100                                        ______________________________________                                         *L929: purified natural MCSF from L929 fibroblasts                            **rh: recombinant human MCSF-(3-153) (E. coli 3153!MCSF)                 

B. M-CSF regulates production of IL-1 inhibitor

Macrophages obtained as set forth above in Example 1 were pretreated for24 hours with either culture medium or rh-M-CSF-(3-153), i.e.,recombinant M-CSF {E. coli 3-153!M-CSF} at concentrations of 0.01, 0.03,0.1, 0.3 and 1.0 μg/ml. Cells that were pretreated with medium were thentreated with either medium (to obtain a control), 1.0 μg/ml of LPSalone, rh-M-CSF-(3-153) alone at concentrations of 1.0, 0.3, 0.01, 0.03μg/ml, or with 1.0 μg/ml of LPS with the four concentrations ofrh-M-CSF-(3-153). Cells that were pretreated with rh-M-CSF-(3-153) weretreated with either medium or LPS at 1.0 μg/ml.

To assess the effect on the production of bioactive IL-1, the culturesupernatant was subjected to a thymocyte assay as described above inExample 1 except that results were set forth as a stimulation index. Thestimulation index refers to the cpm in the presence of ConA andmacrophage conditioned medium at a given dilution/cpm in the presence ofConA only.

As indicated in Table 3, the stimulation index values for the controlsamples were 2.5 (for pretreatment and treatment with medium) and 1.19to 1.67 (for pretreatment with varying concentrations of M-CSF andtreatment with medium). The stimulation index value from LPS treatmentWas almost ten times greater, at 20.3. In contrast, in samplespretreated with M-CSF and stimulated with LPS, the stimulation indexvalues ranged only from 6.02 to 10.09, representing an inhibition ofIL-1 bioactivity of 51 to 70%.

Similarly, in samples co-treated with varying concentrations of M-CSF,the stimulation index values ranged from 5.65 to 11.8, representing aninhibition of IL-1 bioactivity of 72 to 42%. The inhibition appeared tobe concentration dependent because the greatest inhibition was observedwith 1.0 μg/ml of M-CSF and the lowest inhibition with 0.03 μg/ml ofM-CSF. The EC₅₀ (effective concentration that results in 50% inhibition)for rh-M-CSF-(3-153) was determined to be approximately 1.1 nM.

                                      TABLE 3                                     __________________________________________________________________________    Effect of recombinant M-CSF on Bioactive IL-1 Production in Response to       LPS                                                                           Pre-                      Stimulation Index                                                                      % Inhibition                               treatment   Treatment     Over ConA control                                                                      of IL-1                                    (24 h)                                                                             Concentration                                                                        (24 h) Concentration                                                                        in Thymocyte Assay                                                                     Bioactivity                                __________________________________________________________________________    Medium                                                                             --     Medium        2.5                                                 Medium                                                                             --     LPS    1 μg/ml                                                                           20.3                                                Medium                                                                             --     M-CSF  1 μg/ml                                                                           1.01                                                Medium                                                                             --     LPS + M-CSF                                                                          1 μg/ml                                                                           5.65     72                                         Medium                                                                             --     M-CSF  0.3 μg/ml                                                                         1.62                                                Medium                                                                             --     LPS + M-CSF                                                                          1 + 0.3 μg/ml                                                                     6.89     66                                         Medium                                                                             --     M-CSF  0.01 μg/ml                                                                        1.61                                                Medium                                                                             --     LPS + M-CSF                                                                          1 + 0.01 μg/ml                                                                    7.78     60                                         Medium                                                                             --     M-CSF  0.03 μg/ml                                                                        1.26                                                Medium                                                                             --     LPS + M-CSF                                                                          1 + 0.03 μg/ml                                                                    11.8     42                                         M-CSF                                                                                1 μg/ml                                                                         Medium --     1.19                                                M-CSF                                                                                1 μg/ml                                                                         LPS    1 μg/ml                                                                           7.3      64                                         M-CSF                                                                               0.3 μg/ml                                                                        Medium --     1.67                                                M-CSF                                                                               0.3 μg/ml                                                                        LPS    1 μg/ml                                                                           6.02     70                                         M-CSF                                                                               0.1 μg/ml                                                                        Medium --     1.68                                                M-CSF                                                                               0.1 μg/ml                                                                        LPS    1 μg/ml                                                                           6.04     70                                         M-CSF                                                                              0.03 μg/ml                                                                        Medium --     1.43                                                M-CSF                                                                              0.03 μg/ml                                                                        LPS    1 μg/ml                                                                           7.8      62                                         M-CSF                                                                              0.01 μg/ml                                                                        Medium --     1.51                                                M-CSF                                                                              0.01 μg/ml                                                                        LPS    1 μg/ml                                                                           10.9     51                                         __________________________________________________________________________

A similar experiment was conducted with varying concentrations ofnatural M-CSF purified from L929 cells. As set forth in Table 3a,pretreatment with M-CSF inhibited the bioactivity of IL-1 produced frommacrophages in response to LPS 39 to 93%. As above, the greatestinhibition was observed with the largest concentration of M-CSF. TheEC₅₀ for L929 M-CSF was found to be approximately 0.6 nM.

                                      TABLE 3a                                    __________________________________________________________________________    Effect of Purified L929 M-CSF on Bioactive IL-1 Production                    Pre-                                                                          treatment                                                                          Concentration                                                                        Treatment                                                                          Concentration                                                                        Stimulation Index                                                                     % Inhibition                                  __________________________________________________________________________    Medium                                                                             --     Medium                                                                             --     1.21    --                                            Medium                                                                             --     LPS  1 μg/ml                                                                           8.53    --                                            L929  10 Ku/ml                                                                            Medium                                                                             --     1.75    --                                            L929  10 Ku/ml                                                                            LPS  1 μg/ml                                                                           2.43    93                                            L929 3.3 Ku/ml                                                                            Medium                                                                             --     1.45                                                  L929 3.3 Ku/ml                                                                            LPS  1 μg/ml                                                                           4.21    62                                            L929 1.1 Ku/ml                                                                            Medium                                                                             --     1.49                                                  L929 1.1 Ku/ml                                                                            LPS  1 μg/ml                                                                           5.97    39                                            __________________________________________________________________________

These two experiments were repreated more than twelve times with variouspreparations of M-CSF and each experiment yielded similar results. Thus,these experiments indicate that either pretreatment or cotreatment withM-CSF decreases the bioactivity of IL-1 in response to LPS. In addition,that effect appears to be concentration dependent.

These results are set forth graphically in FIG. 3. When the bioactivityof IL-1 (CPM/1000) is plotted against the pretreatment and treatmentregimens, the greatest increase in bioactive IL-1 is seen withpretreatment with medium and treatment with LPS (C-L) as compared withpretreatment and treatment with media (C--C). A much more modestincrease in bioactive IL-1 is seen With pretreatment with medium andtreatment with a combination of LPS and M-CSF (C-LM). A drop inbioactive IL-1 is seen with samples pretreated with M-CSF and treatedwith LPS (M-L). Thus, it appears that M-CSF regulates the production ofIL-1 in macrophages stimulated by LPS.

C. Potential mechanism of action of M-CSF on IL-1 bioactivity

Is set Forth above in FIGS. 1 and 2, LPS induces the expression of IL-1(primarily IL-1α in macrophages) and as set forth in FIG. 3 and Tables 3and 3a above, LPS also increases the production of bioactive IL-1. Whencells are treated with LPS and either pretreated or cotreated withM-CSF, the level of bioactive IL-1 decreases. The expression of IL-1,however, does not appear to be affected by cotreatment with LPS andM-CSF. As set forth in FIG. 2, the combination of M-CSF and LPSexhibited a pattern in the autoradiograph of IL-1 expression that wasvery similar to the pattern observed with LPS alone. These observationscould be explained by the M-CSF dependent stimulation of an IL-1inhibitor which in turn decreases the bioactivity of IL-1. Theproduction of an IL-1 inhibitor was tested by conducting an IL-1thymocyte assay which has been supplemented by the addition of both ConAand IL-1. The addition of these two components allows an assessment ofthe action of an IL-1 inhibitor by providing a detectable level ofbioactive IL-1 so that any decrease in the level of IL-1 can bemeasured.

Six sets of conditioned media were added to the IL-1 thymocyte assay asset forth in FIG. 4. In the set pretreated and treated with media(C--C), the level of bioactive IL-1 remained the same. In contrast,although some decrease in bioactive IL-1 was seen with sets pretreatedwith media or M-CSF and treated with M-CSF or LPS or a combination ofM-CSF and LPS, the only real decrease in IL-1 bioactivity was seen inthe set pretreated with M-CSF and treated with LPS (M-L). Thus, thisstudy implicates M-CSF in the production of an inhibitor to IL-1.

To determine whether the inhibitor was specific to IL-1, a similarexperiment was conducted with IL-6. Six sets of macrophages werepretreated with either media (C) or M-CSF (M) and treated with eithermedia, M-CSF, LPS, or a combination of M-CSF and LPS as set forth inFIG. 5. The activity of IL-6 was assessed using the IL-6 B-9 assay setforth above.

As demonstrated in FIG. 5, a similar pattern of IL-6 bioactivity wasobserved in cells pretreated with media and treated with LPS, in cellspretreated with media and treated with a combination of LPS and M-CSF,and in cells pretreated with M-CSF and treated with LPS. The inhibitorstimulated by M-CSF appeared to have no effect on IL-6 activity. Thispattern is quite different from the pattern observed with IL-1production in FIG. 4 in which a dramatic decrease in IL-1 was seen inculture supernatant from cells pretreated with M-CSF and treated withLPS. Thus, this study indicates that the IL-1 inhibitor is specific toIL-1 and that, under the same conditions, M-CSF does not regulate theproduction of an inhibitor specific to IL-6.

EXAMPLE 3 Injection of M-CSF in vivo results in reduction of IL-1bioactivity produced in response to LPS

Eight inbred female pathogen-free C57B1/6 mice (Charles River BreedingLaboratories, Wilmington, Mass.) were injected intraperitoneally with1.0 ml of sterile Brewer's thioglycollate (TG) at time 0 and dividedinto four groups. As set forth in Table 4 below, at 72 hours, the micein group 1, the control group, and group 2 were pretreated with 0.2 mlphosphate buffered saline (hereinafter "PBS") and the mice in groups 3and 4 were pretreated with PBS containing M-CSF-(3-153) at 4 μg/mouse.At 90 hours, the control group 1 was treated with PBS, group 2 wastreated with PBS containing 50 μg/mouse of LPS (to show the effect ofLPS treatment), group 3 was treated with PBS (to show the effect ofM-CSF pretreatment alone), and group 4 was treated with PBS containing50 μg/mouse of LPS (to show the effect of M-CSF pretreatment on LPStreatment).

Eight hours later, the mice were sacrificed and their peritoneal cellscollected as set forth above in Example 1. Cells at 1×10⁶ cells/wellwere plated in Costar plates and treated with conditioned medium.

Twenty hours later, the conditioned media was collected and titratedinto the IL-1 bioassay set forth above (thymocyte pulsed with ConA).Results are expressed as a mean cpm of triplicate wells.

The percent inhibition of IL-1 bioactivity was determined as follows:##EQU1##

As set forth below in Table 4, at 1/4 dilution of conditioned media,treatment with LPS (group 2) yielded 13103 CPM of bioactive IL-1 whilepretreatment with M-CSF (group 4) yielded 5050 CPM. At 1/12 dilution,treatment with LPS (group 2) yielded 5385 CPM compared to 2592 CPMobtained with pretreatment with M-CSF (group 4). Thus, at both 1/12 and1/4 dilutions of the conditioned media, pretreatment with M-CSFinhibited the bioactivity of IL-1 produced in response to LPS by 69.9%,indicated that M-CSF can inhibit IL-1 bioactivity both in vitro and invivo.

                  TABLE 4                                                         ______________________________________                                        Injection of M-CSF In Vivo Results in Reduction                               of IL-1 Bioactivity Produced in Response to LPS                                               Dilution of                                                                   Conditioned Media                                             Group Treatment of Mice in Vivo                                                                     1/4      1/12  % Inhibition                             ______________________________________                                        1     TG (0 hrs) →                                                                           1583     1353  --                                             PBS (72 hrs) →                                                         PBS (90 hrs)                                                            2     TG (0 hrs) →                                                                           13103    5385  --                                             PBS (72 hrs) →                                                         LPS (90 hrs)                                                            3     TG (0 hrs) →                                                                           1301     1308  --                                             M-CSF (72 hrs) →                                                       PBS (90 hrs)                                                            4     TG (0 hrs) →                                                                           5050     2592  69.9                                           M-CSF (72 hrs) →                                                       LPS (90 hrs)                                                            ______________________________________                                    

EXAMPLE 4 Separation and assessment of the IL-1 inhibitor A. Separationand Characterization of the IL-1 inhibitor

To further assess the IL-1 inhibitor, the conditioned media treated withM-CSF and LPS was subjected to a QAE-52 column to separate the IL-1 fromthe IL-inhibitor. As an anion exchanger, QAE-52 at pH 7.4 has thecapability to separate IL-1α, which has a pI of 5.5, from the IL-1inhibitor.

To separate the IL-1 inhibitor, 3.0×10⁸ peritoneal exudate cells werecultured in 100 mm tissue culture dishes at a cell density of 2.3×10⁵/cm² (approximately 1.8×10⁷ cells/plate) in complete medium. After 2.0hours incubation, the plates were washed twice with complete medium and5.0 ml of M-CSF-(3-153) (1.0 μg/ml) were added per plate and incubatedfor 20 hours. Following this pretreatment step, the fluid was aspirated,plates were washed twice, and 5.0 ml of RPMI 1640 containing 0.5 mg/mlof bovine serum albumin and 1.0 pg/ml of LPS were added per plate. Theplates were cultured for 40 hours and the conditioned medium wascollected, centrifuged at 1000×g for 15 min. at 4° C. The resultingpellet was diluted with an equal volume of ice cold 20 mM HEPES buffer(pH 7.4). The material was then loaded onto a 1.5×10 cm² column packedwith quaternary ammonium cellulose (QAE-52, Whatman).

The sample that did not attach to the QAE-52 flowed through the columnand was collected and designated "flow thru". The sample that did attachto the QAE-52 was released in the presence of 1 M NaCl and was collectedand designated "eluate". Both flow thru and eluate fractions werediafiltered and concentrated to the same volume in an Amicon chamberover a membrane with a 10 kd cut-off point.

The flow thru and eluate samples were subjected to the IL-1 thymocyteassay, the IL-1 radioreceptor assay, a TNF bioassay and an IL-2proliferation assay.

1. IL-1 thymocyte assay

The flow thru and eluate samples were assayed with the IL-1 thymocyteassay set forth above to determine levels of bioactive IL-1 as comparedto the activity of a sample of IL-1 introduced to the assay. The resultsare set forth in FIG. 6.

The eluate exhibited IL-1 activity even greater than that of the IL-1sample since the thymocytes were supplemented with submaximal dose ofIL-1. In contrast, the flow thru exhibited a dramatic decrease in IL-1activity. This indicated that the eluate, which exhibited IL-1 activity,contained IL-1 and perhaps IL-6 and that the flow thru, which decreasedIL-1 activity, contained the IL-1 inhibitor.

2. The IL-1 radioreceptor assay

Both the flow thru and eluate samples were assayed with the IL-1radioreceptor assay set forth in Example 1. As set forth in FIG. 7, bothfractions were able to displace ¹²⁵ IL-1α bound to EL-4.6 cells althoughthe flow thru exhibited greater displacement than did the eluate. Thissuggested that both the IL-1 and the IL-1 inhibitor bound to the IL-1receptor.

3. The TNF assay

To determine the specificity of the IL-1 inhibitor present in the flowthru of the QAE-52, the flow thru sample was tested in the TNF bioassaydescribed by Nedwin, G. E., et al., "Effect of interleukin 2, interferongamma, and mitogens on the production of tumor necrosis factors α andβ", J. Immunology 135: 2492 (1985). In this assay, 5.0×10⁴ cells perwell of L929 fibroblasts were plated in flat-bottomed microtiter platesin the presence of 1.0 U/ml of actinomycin D. Diluted samples from theQAE-52 flow thru were added with or without serial dilutions of TNF-α(obtained from Amgen) to a final volume of 0.2 ml. Following 24 hours ofincubation at 37° C., under 6.0% CO₂, the monolayers were washed. Celllysis was determined by the addition of a solution of 0.5% (w/v) crystalviolet in a mixture of methanol and water (1:4 (v/v)) and quantitated bythe measurement of absorbance at 570 nm on a Titertek multiscan ELISAreader.

As shown in FIG. 8, the curves corresponding to the medium and the 12.5%flow thru exhibit virtually identical patterns, indicating that theQAE-52 flow thru neither inhibits nor stimulates the ability of TNF-α todestroy L929 indicator cells. Thus, this study indicates that the IL-1inhibitor is specific to IL-1 and has no effect on TNF activity.

4. The IL-2 bioassay

To determine the specificity of the IL-1 inhibitor in the QAE flow thru,both the QAE flow thru and the QAE eluate were tested in a bioassay forIL-2 described in Gillis, S, et al., "T cell growth factor parameters ofproduction and a quantitative microassay for activity", J. Immunol. 120:2027 (1978). As set forth in FIG. 9, neither the flow thru nor theeluate were able to efect the activity of IL-2. Both samples exhibitedan almost straight horizontal line, indicating no promotion orinhibition of IL-2 proliferation.

B. Purification of the IL-1 inhibitor

After determining that the IL-1 inhibitor could resist acid and heat butnot trypsinization, the IL-1 inhibitor was further purified on reversephase-HPLC using acetonitrile as an eluant for C-4 column.

In this purification, the material in the flow thru collection waslyophilized, resuspended in 10% (v/v) acetonitrile/0.1% (v/v)trifluoroacetic acid/water and injected into a C-4 column (The NestGroup) connected to a reversed phase HPLC. An acetonitrile gradient(25%-50% (v/v) over 60 min., with a flow rate of 1.0 ml/min.) wasapplied and fractions were collected, lyophilized, and resuspended inthe corresponding buffer. The samples were tested in the IL-1 thymocytebioassay described above, the IL-1 receptor assay described in Example 1and in the thymocyte assay described in Example 2.

As shown in FIG. 10, the peak activity for IL-1 inhibition as determinedby IL-1 bioassay (the solid lines) is found in fractions 13-38. Thosefractions correspond to approximately 37% acetonitrile. Additional testswith the IL-1 radioreceptor binding assay showed that only thesefractions were able to displace IL-1 (the * line). Similarly, in theIL-1 thymocyte assay stimulated with ConA and IL-1 as set forth above,only these fractions were able to inhibit thymocytes proliferation (thetriangles lines). Thus, the IL-1 inhibitor stimulated by M-CSF can beseparated on reverse phase HPLC.

Hereinafter, the anti-allergic composition of the invention will bedescribed in greater detail with reference to Reference Examples thatshow the preparation of the M-CSF derivatives of the invention andExamples that show their anti-allergic properties.

Herein, the human M-CSF derivatives produced are defined as follows.That is to say, the M-CSF produced by an expression vector coding forthe amino acid sequence of Val at position 3 to Thr at position 153 ofthe amino acid sequence defined by the formula (1) with E. coli as thehost is designated "E. coli 3-153!M-CSF" and, similarly, the oneproduced by an expression vector coding for the amino acid sequence ofVal at position 3 to Pro at position 214 is designated "E. coli3-214!M-CSF".

Further, the M-CSF produced by an expression vector coding for the aminoacid sequence of Ser at position 4 to Thr at position 153 with E. colias the host is designated "E. coli 4-153!M-CSF", and similarly oneproduced by an expression vector coding for the amino acid sequence ofSer at position 4 to Pro at position 214 is designated "E. coli4-214!M-CSF".

The M-CSF derivative obtained in Reference Example 1 as produced by anexpression vector coding for a signal peptide comprising a sequence of32 amino acids and the mature human M-CSF protein comprising a sequenceof 522 amino acids with CHO cells as the host is designated "CHO-32-522!M-CSF".

The samples obtained in the examples were assayed for CSF activity bythe following method.

Method of determining CSF activity

Fetal calf serum (FCS, 20 ml), 30 ml of α-medium and 20 ml of α-mediumof 2-fold concentration are mixed together, and the solution ismaintained at 37° C. A 23.3-ml portion of the solution is admixed with10 ml of 1% solution of agar (product of Difco Laboratories) alreadymaintained at 50° C. and the mixture is maintained at 37° C.

Separately, bone marrow cells (BMC) collected from the femur of a BALB/cmouse are washed twice with Hanks' solution and then suspended inα-medium to a concentration of 10⁷ cells/ml, and 1 ml of the suspensionis added to the agar medium maintained at 37° C. The mixture isthoroughly stirred and then maintained at 37° C. A 0.5-ml portion of themixture is placed in each well (Tissue Culture Cluster 12, product ofCostar Corporation) already containing 50 μl of a test sample, and theresulting mixture is quickly stirred and then allowed to stand at roomtemperature. On solidification of the agar in each well, the wells areplaced in a carbon dioxide gas incubator and further incubated at 37° C.for 7 days.

The number of colonies thus produced is counted under a stereoscopicmicroscope to provide an index for the CSF activity. The CSF activity inunits (U/ml) is the value calculated from the above-mentioned colonycount according to the following formula (a).

    CSF activity in units (U/ml)=(colony count)×(dilution factor)÷1.5(a)

When observed morphologically and enzymochemically, the colonies thatformed in the above were mostly macrophage colonies.

Reference Example 1

Preparation of CHO -32-522!M-CSF

Using the culture supernatant of the CHO cell clone No. 2, 3-8Unexamined Japanese Patent Publication HEI 1-104176!microcarrier-cultured in OPTI-MEM (product of Gibco Laboratories)containing 1% FCS, the desired homogeneous CHO -32-522!M-CSF wasobtained by the following purification procedure. In the followingprocedure, the desired protein was detected by the Western blottingmethod. Said Western blotting was performed using Bio-Rad LaboratoriesTransblot cell. The nitrocellulose membrane after transfer was blockedwith PBS containing 1% skim milk, then reacted with a rabbit antiserumagainst M-CSF and further reacted with peroxidase-labeled goatanti-rabbit antibody (product of Bio-Rad Laboratories). For detectingthe M-CSF band, the thus-obtained nitrocellulose membrane was reactedwith a solution of the chromogenic substrate 4-chloro-1-naphthol.

(1) ConA-Sepharose chromatography

The above-mentioned CHO cell culture supernatant (69.3 l) wasconcentrated by ultrafiltration, and a 650-ml portion of the concentrate(842 ml) was used as the starting material and subjected tofractionation with ammonium sulfate. The precipitate fractions obtainedby 35%-65% saturation with ammonium sulfate were dissolved in distilledwater (1,120 ml) to give a sample solution.

Separately, a column (5×25 cm) packed with about 500 ml ofConA-Sepharose gel was equilibrated with 20 mM sodium phosphate buffer(pH 7.4) containing 0.15M NaCl, and the above sample solution wasapplied to the column. After thorough washing with the same buffer,elution was carried out with the same buffer containing 0.5M methylα-D-mannoside. The whole eluate was concentrated by ultrafiltrationusing a YM-10 membrane, then buffer was exchanged to 20 mM sodiumphosphate buffer (pH 7.4), and the resulting solution was subjected, in7 aliquots, to anion exchange high-performance liquid chromatographyunder the following conditions.

(2) Anion exchange high-performance liquid chromatography

Column: TSKgel DEAE-5PW (21.5 mm I.D.×15 cm, product of TosohCorporation)

Eluent A: 40 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM Sodium phosphate buffer (pH 7.4) containing 1.0M NaCl

Flow rate: 3.0 ml/min.

Fraction volume: 6 ml/tube/2 min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      10     0                                                                      20     10                                                                     95     30                                                                    100     100                                                                   110     100                                                                   115     0                                                                     130     0                                                     ______________________________________                                    

As a result of the above elution, the desired M-CSF was eluted intofractions Nos. 26-38 (0.18 to 0.25M NaCl). Said active fractions werepooled, concentrated by ultrafiltration (using a YM-10 membrane) andpurified as follows.

(3) Gel filtration high-performance liquid chromatography

The concentrated sample obtained by the above procedure (2) wassubjected, in 5 aliquots, to gel filtration high-performance liquidchromatography under the following conditions.

Column: TSKgel G3000SWG (21.5 mm I.D.×60 cm, product of TosohCorporation)

Eluent: 50 mM sodium phosphate buffer (pH 7.4) containing 0.3M NaCl

Flow rate: 3.0 ml/min.

Fraction volume: 6 ml/tube/2 min.

As a result of the above gel filtration high-performance liquidchromatography, M-CSF was detected in fractions Nos. 20-27, of whichfractions Nos. 24-27 were pooled, concentrated by ultrafiltration andsubjected to the following purification procedure.

(4) TSKgel Ether-5PW high-performance liquid chromatography

The concentrated sample (12 ml) obtained by the above procedure (3) waspurified, in 8 aliquots, under the following conditions.

Prior to injection into the column, the above-mentioned concentrate wasadmixed with 80% ammonium sulfate-saturated 20 mM sodium phosphatebuffer (pH 7.4) and then subjected to chromatography.

Column: TSKgel Ether-5PW (7.5 mm I.D.×75 mm, product of TosohCorporation)

Eluent A: 20 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 40% Ammonium sulfate-saturated 20 mM sodium phosphate (pH 7.4)

Flow rate: 1.0 ml/min.

Fraction volume: 2 ml/tube/2 min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      100                                                                    5      100                                                                   65       0                                                                    80       0                                                                    85      100                                                                   100     100                                                   ______________________________________                                    

As a result of the above chromatography M-CSF was detected in fractionsNos. 15-18 (22%-17% saturated ammonium sulfate concentrations).Fractions having the same fraction number were respectively pooled andeach pool was subjected, in 3 or 4 aliquots, to reversed-phasehigh-performance liquid chromatography.

(5)TSKgel Phenyl-5PWRP reversed-phase high-performance liquidchromatography

Column: TSKgel Phenyl-5PWRP (7.5 mm I.D.×75 mm, product of TosohCorporation)

Eluent A: 0.1% TFA

Eluent B: n-Propanol: 1% TFA (9:1)

Flow rate: 0.8 ml/min.

Fraction volume: 1.6 ml/tube/2 min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      5      0                                                                     10      25                                                                    50      45                                                                    60      100                                                                   70      100                                                                   75      0                                                                     95      0                                                     ______________________________________                                    

As a result of the above chromatography, M-CSF was detected in twoconsecutive fractions out of fractions Nos. 5-8 (32%-34% n-propanol),although the fraction numbers of the two varied a little depending onthe sample and experiment.

The fractionation was carried out using tubes already containing 200 μMdisodium phosphate per tube. The M-CSF fractions were concentrated todryness by centrifugation tube by tube on a concentrator (product ofTomy Seiko Co., Ltd.) and dissolved in 500 μl per tube of distilledwater and the resulting solutions were subjected to experimentation.

(6) SDS-PAGE of CHO -32-522!M-CSF

According to the method of Laemmli Laemmli, U. K., Nature, 277, 680(1970)!, the CHO -32-522!M-CSF obtained by the above procedure (5) wasadmixed with Laemmli's sample buffer either of the one containing2-mercaptoethanol (2-ME⁺) and the one free of it (2-ME³¹ )!, therespective mixtures were heat-treated at 95° C. for 10 minutes and thensubjected to SDS-PAGE using mini slab gels (gel concentration 12%).Prestained markers (product of Bio-Rad Laboratories) were used asmolecular weight markers. For staining, Silver Stain (product of WakoPure Chemical Industries) was used.

As a result, under non-reducing conditions (state of 2-ME⁻), a smearedband was detected over the molecular weight range of 62,000-115,000 witha main component at a molecular weight of about 85,000 and, underreducing conditions (state of 2-ME⁺), a smear band was detected over39,000-46,000 with a main component at 43,000.

(7) N-Terminal region amino acid sequence of CHO -32-522!M-CSF

The N-terminal region amino acid sequence of the CHO -32-522!M-CSFobtained by the above procedure (5) was determined using a gaseous phasesequencer (product of Applied Biosystems).

As a result, the sequence of the N-terminal 10 amino acids was confirmedto be as follows. The amino acid (X') in cycle 7 could not be identifiedbut was estimated as Cys based on the gene structure.

Glu-Glu-Val-Ser-Glu-Tyr-X'-Ser-His-Met-

Reference Example 2

1! Preparation of plasmid ptrpIL-2X-M-CSF101

The plasmid pcDM-CSF11-185 prepared from the plasmid pcDM-CSF11containing the M-CSF gene (λcM11 cDNA, about 2.5 kb) (cf. UnexaminedJapanese Patent Publication HEI 1-104176)! was digested with therestriction enzymes ScaI and BamHI, and a ScaI-BamHI DNA fragment (about450 bp) was isolated and purified by agarose gel electrophoresis. Then,the synthetic linker (A) shown below was ligated to the ScaI cleavagesite of the above-obtained DNA fragment using T4 DNA ligase to give anXbaI-BamHI DNA fragment (about 480 bp) having an XbaI (restrictionenzyme) cleavage site on the ScaI cleavage end side.

Synthetic linker (A): ##STR2##

The thus-obtained DNA fragment was inserted into the human IL-2expression plasmid ptrpIL-2D8Δ (Unexamined Japanese Patent PublicationSHO 63-12958) between the XbaI and BamHI cleavage sites, whereby thedesired plasmid ptrpIL-2X-M-CSF101 was obtained.

A transformant obtained by transforming Escherichia coli HB101 with saidplasmid has been deposited, under the name of Escherichia coliHB101/ptrpIL-2X-M-CSF101, in the Fermentation Research Institute, Agencyof Industrial Science and Technology with the deposition number of FERMBP-2226 (E. coli 3-153! FERM BP-2226! since Dec. 26, 1988.

2! Isolation and purification of E. coli 3-153! M-CSF

(1) Preparation of M-CSF fraction from Escherichia coli

To 1.5 g (wet weight) of an E. coli strain SG21058 harboring the plasmidptrpIL-2X-M-CSF101 obtained by the above procedure 1! was added 50 ml of50 mM Tris-hydrochloride buffer (pH 7.0) containing 0.5M sucrose, andthe mixture was thoroughly stirred. Then, 6 ml of 2 mg/ml lysozyme wasadded, 4 ml of 0.14M EDTA was further added and, after 15 minutes ofstirring at 4° C., the mixture was centrifuged at 10,000revolutions/minute for 20 minutes.

The supernatant was discarded, and the pellet was washed with the samebuffer (50 mM Tris-hydrochloride containing 0.5M sucrose, pH 7.0) andsubjected to centrifugation, which was carried out similarly at 10,000revolutions/minute for 20 minutes and gave spheroplasts as a pellet. Thepellet was suspended in 50 ml of 50 mM Tris-hydrochloride (pH 7.0) andthe suspension was subjected to sonication at 20 KHz for 10 minutes andthen centrifuged at 10,000 revolutions/minute for 20 minutes. The pelletwas washed with the same buffer (50 mM Tris-hydrochloride, pH 7.0) andagain centrifuged under the same conditions to give an M-CSF fraction asa pellet.

(2) Refolding of M-CSF from M-CSF fraction

To the M-CSF fraction obtained by the above procedure (1) was added 100ml of 50 mM Tris-hydrochloride (pH 7.0) containing 7.0M guanidinehydrochloride, and the mixture was solubilized by agitating with astirrer at 4° C. for 1 hour. This solution was slowly added dropwiseinto a beaker already containing 300 ml of 10 mM Tris-hydrochloride (pH8.5) (agitated with stirrer) and, after completion of the dropping, themixture was thoroughly dialyzed against 10 mM Tris-hydrochloride (pH8.5) at 4° C. and then centrifuged at 10,000 revolutions/minute for 20minutes. The precipitate was discarded and the supernatant wasrecovered.

The refolded M-CSF is present in the thus-obtained supernatant.

(3) Purification of M-CSF

The M-CSF obtained in the above (2) was purified as follows.

(3-1) Gel filtration high-performance liquid chromatography

The supernatant obtained by the above procedure (2) was concentratedwith an ultrafiltration apparatus of (product of Amicon; membrane: YM-10membrane, product of Amicon), and the concentrate was passed through a0.45 μm Millipore filter and then subjected to gel filtrationhigh-performance liquid chromatography under the following conditions.

Column: TSKgel G 3000SW (60 cm×21.5 mm I.D., product of TosohCorporation)

Eluent: 50 mM sodium phosphate buffer (pH 6.8) containing 0.3M NaCl

Flow rate: 3.0 ml/min.

Fraction volume: 6 ml/tube/2 min.

In the above procedure, judging from the elution positions of standardproteins for gel filtration HPLC (product of Oriental Yeast Co., Ltd.),namely glutamate dehydrogenase (molecular weight 290,000), lactatedehydrogenase (molecular weight 142,000), enolase (molecular weight67,000) and adenylate kinase (molecular weight 32,000), the molecularweight of M-CSF was estimated to be 32,000.

The active fractions were collected and buffer exhange was performedagainst 40 mM sodium borate (pH 8.0) by the ultrafiltration apparatusmentioned above.

(3-2) TSKgel DEAE-5PW ion exchange high-performance liquidchromatography

The active eluate fraction obtained by the above procedure (3-1) wassubjected to TSKgel DEAE-5PW ion exchange high-performance liquidchromatography under the following conditions.

Column: TSKgel DEAE-5PW (7.5 mm I.D.×75 mm, product of TosohCorporation)

Eluent A: 40 mM Sodium borate buffer (pH 8.0) containing 5% methanol

Eluent B: 40 mM Sodium borate buffer (pH 8.0) containing 1.0M NaCl and5% methanol

Flow rate: 1.0 ml/min.

Fraction volume: 1.0 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      7      0                                                                     42      30                                                                    47      100                                                                   52      100                                                                   57      0                                                                     62      0                                                     ______________________________________                                    

Judging from the results (elution pattern) of the above TSKgel DEAE-5PWion exchange high-performance liquid chromatography, the peaks observedwith fractions Nos. 42-43 (NaCl concentration 0.23-0.25M) correspondedto M-CSF and said peaks were collected. Thus, purified M-CSF (E.Coli3-153!M-CSF) was obtained from Escherichia coli.

Reference Example 3

Preparation of CHO -32-522!M-CSF

A 930 ml quantity of a concentrated culture supernatant obtained byculturing CHO cells in the same manner as in Reference Example 1 wassubjected to fractionation with ammonium sulfate. Fractions precipitatedby 25-65% saturation with ammonium sulfate were obtained. Then thefractions were dissolved in distilled water (1180 ml) and purified bythe following method. The detection of M-CSF was conducted, as inReference Example 1, by Western blotting.

(1) ConA-Sepharose chromatography

A column (5×25 cm) packed with about 500 ml of ConA-Sepharose gel wasequilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing0.15M NaCl, and the above solution was applied to the column. Afterthorough washing with the same buffer, elution was carried out with thesame buffer containing 0.5M methyl α-D-mannoside. The whole eluate wasconcentrated by ultrafiltration using a YM-10 membrane, then buffer wasexchanged to 20 mM sodium phosphate buffer (pH 7.4), and the resultingsolution was subjected, in 5 aliquots, to anion exchangehigh-performance liquid chromatography.

(2) Anion exchange high-performance liquid chromatography

Column: TSKgel DEAE-5PW (21.5 mm ID×15 cm, product of Tosoh Corporation)

Eluent A: 40 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM Sodium phosphate buffer (pH 7.4) containment 1.0M NaCl

Flow rate: 3.0 ml/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      10     0                                                                      20     10                                                                     95     30                                                                    100     100                                                                   110     100                                                                   115     0                                                                     130     0                                                     ______________________________________                                    

AS a result of the above elution, the desired M-CSF was eluted intofractions Nos. 26-43. Main M-CSF fractions, i.e., fractions Nos. 30-39were pooled, concentrated by ultrafiltration (using a YM-10 membrane)(15 ml) and purified, in 4 aliquots as follows.

(3) TSKgel Phenyl-5PWRP reversed-phase high-performance liquidchromatography

Column: TSKgel Phenyl-5PWRP (25.5 mm ID×15 cm, product of TosohCorporation)

Eluent A: 0.1% TFA

Eluent B: n-Propanol: 1.0% TFA (9:1)

Flow rate: 3 ml/min.

Fraction volume: 1.5 ml/tube/0.5 min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      10     0                                                                      20     20                                                                    100     45                                                                    120     100                                                                   130     100                                                                   140     0                                                                     180     0                                                     ______________________________________                                    

The M-CSF fractions of the above eluted fractions were pooled,concentrated by ultrafiltration (using a YM-10 membrane), furtherconcentrated by Centricon 30 (product of Amicon) (1 ml), and subjected,in 3 aliquots, to gel filtration high performance liquid chromatography.

(4) Gel filtration high-performance liquid chromatography

Column: Superose 12HR 10/30 (10 mm ID×30 cm, product of Pharmacia LKB)

Eluent: 20 mM Sodium phosphate buffer (pH 7.4) containing 0.3M NaCl

Flow rate: 0.8 ml/min.

Fraction volume: 0.8 ml/tube/min.

As a result of the above chromatography, M-CSF was eluted in fractionsNos. 15-17, of which fraction No. 16 was pooled and subjected toexperiment (5.6 mg, specific activity: 3.8×10⁷ units/mg protein).

Reference Example 4

Improved isolation and purification of M-CSF derivative (E. coli3-153!-M-CSF)

(1) Preparation of M-CSF fraction from E. coli

To 7.5 9 (Feet weight) of the E. coli strain SG21058 carrying plasmidptrpIL-2X-M-CSF101 was added 1.0 ml of 50 nM Tris-hydrochloride buffer(pH 7.0), and the mixture was stirred thoroughly. Then, 6 ml of 4 mg/mllysozyme and an amount of EDTA to make a final concentration of 10 mMwere added, and the mixture was stirred at 4° C. for 15 minutes, thensonicated (20 KHz, 10 minutes, 200 W) and further centrifuged at10,000×g/minute for 20 minutes to give a pellet. This was further washedWith a buffer for washing (50 mM Tris-hydrochloride containing 2% TritonX100, pH 7.0) and again subjected to centrifugation under the sameconditions. Two repetitions of this procedure gave an M-CSF fraction(pellet).

(2) Refolding of M-CSF from M-CSF fraction

To the M-CSF fraction obtained by the above procedure (1) was added 20ml of 50 mM Tris-hydrochloride buffer (pH 7.0) containing 7M guanidinehydrochloride and 25 mM 2-mercaptoethanol, and the mixture was stirredat room temperature for at least 4 hours for dissolution. This solutionwas gradually added dropwise into a beaker already containing 2,000 mlof 50 mM Tris-hydrochloride buffer (pH 8.5) containing 0.5 mMreduced-form glutathione, 0.1 mM oxidized-form glutathione and 2M urea(with agitation with a stirrer). Then, the resultant mixture was alloyedto stand at 4° C. for at least 2 days. The solution was then centrifugedat 10,000×g/minute for 30 minutes. The precipitate was removed and thesupernatant was recovered.

The refolded M-CSF occurs in the thus-obtained supernatant.

(3) Purification of M-CSF

The supernatant obtained by the above procedure (2) was purified in thefollowing manner.

(3-1) Concentration by ion exchange chromatography

The refolded product solution obtained by the above procedure (2) wasapplied to a QAE-Zeta Prep 100 (product of Pharmacia-LBK) equilibratedin advance with 50 m Tris-hydrochloride buffer (pH 8.5) and, afterthorough washing with the above-mentioned buffer, the M-CSF fraction waseluted with the above-mentioned buffer containing 0.5M NaCl.

(3-2) Hydrophobic high-performance liquid chromatography Ammoniumsulfate was added to the fraction obtained in the above manner to give a30%-saturated solution and the solution was centrifuged at10,000×g/minutes for 20 minutes. The sediment was removed and thesupernatant was recovered. This supernatant was passed through a 0.45-μmMillipore filter and then subjected to hydrophobic high-performanceliquid chromatography under the following conditions.

Column: TSKgel Phenyl 5PW (21.5 mm I.D.×150 mm, product of TosohCorporation)

Eluent A: 30% Ammonium sulfate-saturated 40 mM sodium phosphate buffer(pH 7.4)

Eluent B: 40 mm Sodium phosphate buffer (pH 7.4)

Flow rate: 3.0 ml/min.

Fraction volume: 3.0 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      7      0                                                                     47      100                                                                   52      100                                                                   57      0                                                     ______________________________________                                    

As a result of the above, an M-CSF activity was eluted in fractionscorresponding to the ammonium sulfate concentrations of 6-3%. Saidactive fractions were collected and subjected to buffer exchange to 40mM sodium phosphate (pH 7.4) using an ultrafiltration apparatus.

(3--3) Anion exchange high-performance liquid chromatography

The fraction obtained by the above procedure (3-2 was subjected to anionexchange high-performance liquid chromatography under the followingconditions.

Column: TSKgel DEAE-5PW (21.5 mm I.D.×150 mm, product of TosohCorporation)

Eluent A: 40 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 40 mM Sodium phosphate buffer (pH 7.4) containing 1.0M NaCl

Flow rate: 3.0 ml/min.

Fraction volume: 3.0 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      5      0                                                                     43      30                                                                    48      100                                                                   53      100                                                                   58      0                                                     ______________________________________                                    

From the result (elution pattern) of the above anion exchangehigh-performance liquid chromatography, the peaks observed withfractions Nos. 35 and 36 (NaCl concentration 0.28-0.29M) were found tocorrespond to the M-CSF. Said peaks were collected and thus the purifiedhuman M-CSF derivative (E. coli 3-153!-M-CSF) of the invention wasobtained.

Reference Example 5

(1) Preparation of ptrpIL-2X-M-CSF201

The plasmid ptrpIL-2X-M-CSF101 was cleaved with the restriction enzymesBstEII and SalI and a 4.7 kb DNA fragment (I) containing the IL-2 andM-CSF DNA portions was obtained.

Separately, plasmid pcDM-CSF11 was cleaved with the restriction enzymeEcoRI. The cleavage ends were rendered blunt-ended using DNA polymeraseI Klenow, fragment and then a SalI linker (5'-pGGTCGACC_(OH) 3')(product of New England Biolabs) was ligated thereto. This ligationproduct was cleaved with the restriction enzymes SalI and BstEII to givea BstEII-SalI fragment (II) of about 1.1 kb.

The above-mentioned DNA fragments (I) and (II) were ligated togetherusing T4 DNA ligase and the ligation product was used to transformcompetent cells of E. coli HB101 to give a desired transformant E. coliHB101 strain carrying the plasmid ptrpIL-2X-M-CSF201.

The thus-obtained plasmid ptrpIL-2X-M-CSF201 encodes two polypeptideswithin the transcription unit under the control of the E. colitryptophan promoter. One is a 65-amino-acid polypeptide composed ofmethionine (translation initiation), amino terminal 60 amino acids ofhuman IL-2 and 4 amino acids encoded by the synthetic DNA linker, andthe other is a polypeptide comprising a human M-CSF derivative composedof Met (translation initiation) and those 520 amino acids covering theposition-3 amino acid (Val) to the position-522 amino acid (Val) of theamino acid sequence defined by the formula (1).

In such two-cistron expression system, the translation of the secondcistron is initiated by binding of a ribosome to the second SD sequencelocated in the synthetic DNA linker.

(2) Preparation of ptrpIL-2X-M-CSF202

The plasmid ptrpIL-2X-M-CSF201 was cleaved with the restriction enzymesNcoI and SalI and an NcoI-SalI DNA fragment of about 4.8 kb wasrecovered. Both ends of this DNA fragment was ligated to each otherusing synthetic oligodeoxynucleotides 5'-CATGGCCTGATAAG-3' and5'-TCGACTTATCAGGC-3'! with T4 DNA ligase. Competent cells of E. coliHB101 were transformed with the ligation product and a desiredtransformant E. coli HB101 strain carrying the plasmidptrpIL-2X-M-CSF202 as obtained.

The ptrpIL-2X-M-CSF202 obtained encodes a polypeptide comprising a humanM-CSP derivative composed or met (translation initiation) and those 182amino acids covering the position-3 amino acid (Val) to the position-84amino acid (Ala) of the amino acid sequence defined by the Formula (1)in the second cistron within the transcription unit under the control ofthe E. coli tryptophan promoter.

(3) Preparation of ptrpIL-2X-M-CSF203

The plasmid ptrpIL-2X-M-CSF201 was cleaved with the restriction enzymesBamHI and SalI and a BamHI-SalI DNA fragment of about 4.9 kb wasrecovered. Both ends of this DNA fragment were ligated to each otherusing synethetic oligodeoxynucleotides 5'-GATCCATGATAAG-3' and5'-TCGACTTATCATG-3' ! with T4 DNA<ligase and the ligation product wasused to transform competent cells of E. coli HB101, whereby a desired E.coli HB101 transformant carrying the plasmid ptrpIL-2X-M-CSF203 wasobtained.

The thus-obtained plasmid ptrpIL-2X-M-CSF203 encodes a polypeptidecomprising a human M-CSF derivative composed of Met (translationinitiation) and those 212 amino acids covering the position-3 amino acid(Val) to the position-214 amino acid (Pro) of the amino acid sequencedefined by the formula (1) in the second cistron within thetranscription unit under the control of the E. coli tryptophan promoter.

The above-mentioned E. coli HB101 strain carrying the plasmidptrpIL-2X-M-CSF203 has been deposited, under the designation"Escherichia coli HB101/ptrpIL-2X-M-CSF203", with the FermentationResearch institute, Agency of Industrial Science and Technology underthe deposition number FIRM P-11053 as of Oct. 18, 1989.

(4) Preparation of ptrpIL-2X-M-CSF204

The plasmid ptrpIL-2X-MS-CSF201 was cleaved with the restriction enzymesSmaI and SalI and a SmaI-SalI DNA fragment of about 5.0 kb wasrecovered. Both ends of this DNA fragment were ligated to each otherusing synthetic oligodeoxynucleotides 5'-GGGTGATAAG-3' and5'-TCGACTTATCACCC-3'! with T4 DNA ligase and the ligation product wasused to transform competent cells of E. coli HB101 to give a desired E.coli HB101 transformant carrying the plasmid ptrpIL-2X-M-CSF204.

The plasmid ptrpIL-2X-M-CSF204 thus obtained encodes a polypeptidecomprising a human M-CSF derivative composed of Met (translationinitiation) and those 256 amino acids covering the position-3 amino acid(Val) to the position-258 amino acid (Gly) of the amino acid sequencedefined by the formula (1) in the second cistron within thetranscription unit under the control of the E. coli tryptophan promoter.

(5) Preparation of ptrpIL-2X-M-CSF205

The plasmid ptrpIL-2X-M-CSF201 was cleaved with the restriction enzymesSphI and SalI and an SphI-SalI DNA fragment of about 5.1 kb wasrecovered. Both ends of this DNA fragment were ligated to each otherusing synthetic oligodeoxynucleotides 5'-CAGTGATAAG-3' and5'-TCGACTTATCACTGCATG-3'! with T4 DNA ligase and the ligation productwas used to transform competent cells of E. coli HB101 to give a desiredE. coli HB101 transformant carrying the plasmid ptrpIL-2X-M-CSF205.

The thus obtained plasmid ptrpIL-2X-M-CSF205 encodes a polypeptidecomprising a human M-CSF derivative composed of Met (translationinitiation) and those 300 amino acids covering the position-3 amino acid(Val) to the position-302 amino-acid (Gln) of the amino acid sequencedefined by the formula (1) in the second cistron Within thetranscription unit under the control of the E. coli tryptophan promoter.

(6) Preparation of ptrpIL-2X-M-CSF206

The plasmid ptrpIL-2X-M-CSF201 was cleaved with the restriction enzymesKpnI and SalI and a KpnI-SalI DNA Fragment of about 5.2 kb wasrecovered. Both ends of this DNA fragment were ligated to each otherusing synthetic oligodeoxynucleotides 5'-CGCCTGATAAG-3' and5'-TCGACTTATCAGGCGGTAC-3'! with T4 DNA ligase and the ligation productwas used to transform competent cells of E. coli HB101 to give a desiredE. coli HB101 transformant carrying the plasmid ptrpIL-2X-M-CSF206.

The thus-obtained plasmid ptrpIL-2X-M-CSF206 encodes a polypeptidecomprising a human M-CSF derivative composed of Met (translationinitiation) and those 332 amino acids covering the position-3 amino acid(Val) to the position-334 amino acid (Ala) of the amino acid sequencedefined by the formula (1) in the second cistron within thetranscription unit under the control of the E. coli tryptophan promoter.

(7) Expression of M-CSF derivatives

The plasmid respectively obtained by the above procedures (1) to (6)were each introduced into the E. coli strain SG2G21058 J. Bacteriol.,164, 1124-1135 (1985)! by the transformation method and transformantsrespectively designated E. coli SG21058/ptrpIL-2X-M-CSF201, E. coliSG21058/ptrpIL-2X-M-CSF202, E. coli SG21058/ptrpIL-2X-M-CSF203, E. coliSG21058/ptrpIL-2X-M-CSF204, E. coli SG21058/ptrpIL-2X-M-CSF205 and E.coli SG21053/ptrpIL-2X-M-CSF206! were obtained.

These transformants were cultivated in L3 medium (T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, p. 440(1982), Cold Spring Harbor Laboratory) containing 50 g/ml ampicillinovernight at 37° C. A 0.5-ml portion of each culture was added to 50 mlof M9 medium (see the reference cited above) supplemented with 1%casamino acid, 5 μg/ml thiamine hydrochloride, 20 μg/ml L-cysteine and50 μg/ml ampicillin with the glucose concentration adjusted to 0.4%, andshake culture was performed at 37° C. for 8 hours. Then cells wererecovered by centrifugation (5,000 rpm), and the protein produced wasanalyzed by SDS-PAGE and Western blotting.

The Western blotting was performed using Bio-Rad's Transblot cells.Nitrocellulose membranes after transfer were blocked with PBS⁻containing 1% bovine serum albumin, then reacted with a rabbit antiserumagainst M-CSF and further reacted with peroxidase-labeled goatanti-rabbit antibody (product of Bio-Rad Laboratories). M-CSF banddetection was performed by reacting the thus-obtained nltrocellulosemembranes with a solution of the chromogenic substrate4-chloro-1-naphthol.

In E. coli SC21058/ptrpIL-2X-M-CSF202, E. coliSG21058/ptrpIL-2X-M-CSF203, E. coli SG21058/ptrpIL-2X-M-CSF204, E. coliSG21058/ptrpIL-2X-M-CSF205 and E. coli SG21058/ptrpIL-2X-M-CSF206, theM-CSFs encoded in the second cistrons were respectively detected atpositions corresponding to the expected molecular weights.

(8) Isolation and purification of M-CSF derivative (E. coli3-2141-M-CSF)

To 10 g (wet weight) of E. coli SC21058 cells harboring the plasmidptrpIL-2X-M-CSF203 obtained by the above procedure (3) was added 50 mMTris-hydrochloride buffer (pH 7.0) to make the whole volume 100 ml,followed by thorough stirring. Then, 4 ml of 6 mg/ml lysozyme was added,8 ml of 0.14M EDTA was further added, and the mixture was stirred at 4°C. for 15 minutes, then sonicated (200 KHz, 2 minutes, 200 W) andfurther centrifuged at 10,000 revolutions/minute for 20 minutes. Thepellet obtained was thoroughly washed with 50 mM Tris-hydrochloride (pH7.0) containing 2% Triton X-100 and again subjected to centrifugationunder the same conditions to give an M-CSF fraction as the pellet.

To the thus-obtained M-CSF fraction was added 20 ml of 50 mMTris-hydrochloride (pH 7.0) containing 7.0M guanidine hydrochloride and50 mM mercaptoethanol, and the mixture was stirred at room temperaturefor 4 hours for reducing, denaturing and dissolving the protein. Thissolution was gradually added dropwise into a breaker already containing2,000 ml of 50 mm Tris-hydrochloride (pH 8.5) containing 0.5 mMreduced-form glutathione, 0.1 mM oxidized-form, glutathione and 2M urea(with agitation with a stirrer) for 100-fold dilution. After completionof the dropping, the dilution was allowed to stand at 4° C. for at least2 days and then centrifuged at 3,000 revolutions/minute for 30 minutes.The pellet was removed and the supernatant (2 liters) was recovered.

The refolded M-CSF exists in the thus-obtained supernatant.

One liter of the centrifugation supernatant obtained by the aboveprocedure was concentrated using an ultrafiltration device (product ofAmicon, with a YM-10 membrane (Amicon)), and the concentrate wascentrifuged at 10,000 revolutions/minute for 20 minutes. The pellet wasremoved and the supernatant was recovered. To this supernatant was addedammonium sulfate in an amount sufficient for 30% saturation. Theresultant mixture was again centrifuged in the same manner and thesupernatant was subjected to hydrophobic interaction chromatographyunder the following conditions.

Column: TSKgel Phenyl 5PW (7.5 mm ID×75 mm, product of TosohCorporation)

Eluent A: 30% ammonium sulfate-saturated 50 mM sodium phosphate buffer(pH 7.4)

Eluent B: 50 mm Sodium phosphate buffer (pH 7.4)

Flow rate: 1.0 ml/min.

Fraction volume: 1 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      7      0                                                                     40      100                                                                   45      100                                                                   50      0                                                                     60      0                                                     ______________________________________                                    

As a result of the above procedure, an M-CSF activity was eluted infractions Nos. 40-42 (ammonium sulfate concentration 8-6% saturation).The active fractions were collected, subjected to buffer exchange for 50mM sodium phosphate (pH 7.4) using the above-mentioned ultrafiltrationdevice, and then subjected to DEAE-5PW ion exchange high-performanceliquid chromatography under the following conditions.

Column: DEAE-5PW (7.5 mm ID×75 mm, product of Tosoh Corporation)

Eluent A: 50 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 50 mM Sodium phosphate buffer (pH 7.4) containing 1.0M NaCl

Flow rate: 1.0 ml/min.

Fraction volume: 1.0 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min)                                                                              B %                                               ______________________________________                                                         0      0                                                                      7      0                                                                     42      30                                                                    47      100                                                                   52      100                                                                   57      0                                                     ______________________________________                                    

From the result (elution pattern) of the above DEAE-5PW ion exchangehigh-performance liquid chromatography, the peaks observed in fractionsNos. 31 and 32 (NaCl concentration 0.18-0.20M) corresponded to M-CSF.Said peaks were collected and a human M-CSF derivative of the presentinvention was obtained.

In the above purification steps, the activity of the human M-CSFderivative of the invention, the protein concentration, the specificactivity and the degree of purification were determined. The results areshown below in Table 5.

                                      TABLE 5                                     __________________________________________________________________________             Volume                                                                             Activity                                                                           Protein                                                                             Specific activity                                                                     Purification                                 Purification step                                                                      (ml) (unit)                                                                             (mg)  (unit/mg protein)                                                                     (fold)                                       __________________________________________________________________________    Refolded solution                                                                      1000 4.62 × 10.sup.7                                                              110   4.20 × 10.sup.5                                                                  1.0                                         30% Ammonium                                                                           45   3.93 × 10.sup.7                                                              10.1  3.89 × 10.sup.6                                                                  9.3                                         Sulfate supernatant                                                           Phenyl 5-PW HPLC                                                                       3.6  1.50 × 10.sup.7                                                              1.82  8.24 × 10.sup.6                                                                 19.6                                         DEAE-5PW HPLC                                                                          2.0  7.84 × 10.sup.6                                                              0.514 1.53 × 10.sup.7                                                                 36.4                                         __________________________________________________________________________

(9) SDS-PAGE of M-CSF derivative (E. coli 3-214!M-CSF) Following themethod of Laemmli Laemmli, U.K., Nature, 277, 680 (1970)!, the samplepurified in the above manner was dissolved in each of Laemmli's samplebuffers 2-ME⁺ and 2-ME⁻ ! and each solution was heat-treated at 95° C.for 5 minutes and then subjected to electrophoresis using a microslabgel (apparatus: product of Marisol Sangyo K.K. or Daiichi Chemical Co.,Ltd.!. Prestained markers (product of Bio-Rad Laboratories) were used asmolecular weight markers and Coomassie Brilliant Blue R-250 was used forstaining.

As a result, the molecular weight of E. coli 3-214!M-CSF was found to beabout 52,500 in the 2-ME⁻ state and about 26,900 in the 2-ME⁺ state andthe electrophoresis gave a single band at each of said positions.

Reference Example 6

(1) Preparation of plasmid ptrpIL-2X-M-CSF109

The plasmid pcDM-CSF11-185 prepared from the plasmid pcDM-CSFllcontaining an M-CSF gene (λcM11 cDNA, about 2.5 kb) (cf. UnexaminedJapanese Patent Publication HEI 1-104176)! was digested smith therestriction enzymes ScaI and BamHI and a ScaI-BamHI DNA fragment (about450 bp) was isolated and purified by agarose gel electrophoresis.

Then, a synthetic linker (B) shown below was ligated to the ScaIcleavage site of the thus obtained DNA fragment using T4 DNA ligase,whereby a XbaI-BamHI DNA fragment (about 480 bp) having an XbaI(restriction enzyme) cleavage site on the ScaI cleavage end side wasobtained. ##STR3##

The thus-obtained DNA fragment was inserted into the human IL-2expression plasmid ptrpIL-2D8Δ (cf. Unexamined Japanese PatentPublication SHO 63-12958) between the XbaI and BamHI cleavage sites togive the desired plasmid, ptrpIL-2X-M-CSF109.

The plasmid ptrpIL-2X-M-CSF109 thus obtained encodes two polypeptideswithin the transcription unit under the control of the E. colitryptophan promoter. One is a 65-amino-acid polypeptide composed of Met(translation initiation), amino terminal 60 amino acids of human IL-2and 4 amino acids resulting from the synthetic linker DNA sequence, andthe other is a polypeptide composed of Met (translation initiation) and150 amino acids comprising the 4-position amino acid (Ser) to the153-position amino acid (Thr) of the amino acid sequence defined by theformula (1).

(2) Preparation of ptrpIL-2X-M-CSF402

The plasmid ptrpIL-2X-M-CSF109 was cleaved with the restriction enzymesBstEII and ScaI and a DNA fragment of about 1.5 kb was recovered.

Separately, the plasmid ptrpIL-2X-M-CSF202 was cleaved in the samemanner with the restriction enzymes BstEII and ScaI and a DNA fragmentof about 3.3 kb was recovered.

Both the above DNA fragment was ligated to each other using T4 DNAligase and the ligation mixture was used to transform competent cells ofE. coli HB101 to give an E. coli HB101 transformant carrying the desiredplasmid ptrpIL-2X-M-CSF402.

The thus-obtained plasmid ptrpIL-2X-M-CSF402 encodes, in the secondcistron within the transcription unit under control of the E. colitryptophan promoter, a polypeptide comprising Met (translationinitiation) and a human M-CSF derivative composed of 181 amino acidsfrom the 4-position amino acid (Ser) to the 184-position amino acid(Ala) of the amino acid sequence defined by the formula (1).

(3) Preparation of ptrpIL-2X-M.-CSF403

The plasmid ptrpIL-2X-M-CSF109 was cleaved with the restriction enzymesBstEII and ScaI and a DNA fragment of about 1.5 kb was recovered.

Separately, the plasmid ptrpIL-2X-M-CSF203 was cleaved in the samemanner with the restriction enzymes BstEII and ScaI and a DNA fragmentof about 3.4 kb was recovered.

Both the above DNA fragments were ligated to each other using T4 DNAligase and the ligation mixture was used to transform competent cells ofE. coli HB101, whereby an E. coli HB101 transformant carrying thedesired plasmid ptrpIL-2X-M-CSF403 was obtained.

The thus-obtained plasmid ptrpIL-2X-M-CSF403 encodes, in the secondcistron within the transcription unit under the control of the E. colitryptophan promoter, a polypeptide comprising a human M-CSF derivativecomposed of Met (translation initiation) and 211 amino acids from the4-position amino acid (Ser) to 214-position amino acid (Pro) of theamino acid sequence defined by the formula (1).

(4) Preparation of ptrpIL-2X-M-CSF404

The plasmid ptrpIL-2X-M-CSF109 was cleaved with the restriction enzymesBstEII and ScaI and a DNA fragment of about 1.5 kb Was recovered.

Separately, the plasmid ptrpIL-2X-M-CSF204 Was cleaved in the samemanner with the restriction enzymes BstEII and ScaI and a DNA fragmentof about 3.5 kb was recovered.

Both the above DNA fragments were ligated to each other using T4 DNAligase and the ligation mixture was used to transform competent cells ofE. coli HB101, whereby an E. coli. B101 transformant carrying thedesired plasmid ptrpIL-2X-M-CSF404 was obtained.

The thus-obtained plasmid ptrpIL-2X-M-CSF404 encodes, in the secondcistron within the transcription unit under the control of the E. colitryptophan promoter, a polypeptide comprising a human M-CSF derivativecomposed of Met (translation initiation) and 255 amino acids from the4-position amino-acid (Ser) to the 258-position amino acid (Gly) of theamino acid sequence defined by the formula (1).

(5) Preparation of ptrpIL-2X-M-CSF405

The plasmid ptrpIL-2X-M-CSF109 was cleaved with the restriction enzymesBstEII and ScaI and a DNA fragment of about 1.5 kb was recovered.

Separately, the plasmid ptrpIL-2X-M-CSF205 was cleaved in the samemanner with the restriction enzymes BstEII and ScaI and a DNA fragmentof about 3.6 kb was recovered.

Both the above DNA fragments were ligated to each other using T4 DNAligase and the ligation mixture was used to transform competent cells ofE. coli HB101, whereby an E. coli HB101 transformant carrying thedesired plasmid ptrpIL-2X-M-CSF405 was obtained.

The thus-obtained plasmid ptrpIL-2X-M-CSF405 encodes, in the secondcistron within the transcription unit under the control of the E. colitryptophan promoter, a polypeptide comprising a human M-CSF derivativecomposed of Met (translation initiation) and 299 amino acids from the4-position amino acid (Ser) to the 302-position amino acid (Gln) of theamino acid sequence defined by the formula (1).

(6) Preparation of ptrpIL-2X-M-CSF406

The plasmid-ptrpIL-2X-M-CSF109 was cleaved with the restriction enzymesBstEII and ScaI and a DNA fragment of about 1.5 kb was recovered.

Separately, the plasmid ptrpIL-2X-M-CSF206 was cleaved in the samemanner with the restriction enzymes BstEII and ScaI and a DNA fragmentof about 3.7 kb was recovered.

Both the above DNA fragments were ligated to each other using T4 DNAligase and the ligation mixture was used to transform competent cells ofE. coli HB101, whereby an E. coli HB101 transformant carrying thedesired plasmid ptrpIL-2X-M-CSF406 was obtained.

The thus-obtained plasmid ptrpIL-2X-M-CSF406 encodes, in the secondcistron within the transcription unit under the control of the E. colitryptophan promoter, a polypeptide comprising a human M-CSF derivativecomposed of Met (translation initiation) and 331 amino acids from the4-position amino acid (Ser) to the 334-position amino acid (Ala) of theamino acid sequence defined by the formula (1).

(7) Expression of M-CSF derivatives

The plasmids respectively obtained by the above procedures (1) to (6)were each introduced into the E. coli strain SG21058 J. Bacteriol., 164,1124-1135 (1985)! by the transformation method to give transformantsrespectively designated E. coli SG21058/ptrpIL-2X-M-CSF109, E. coliSG21058/ptrpIL-2X-M-CSF402, E. coli SG21058/ptrpIL-2X-M-CSF403, E. coliSC21058/ptrpIL-2X-M-CSF404, E. coli SG21058/ptrpIL-2X-M-CSF405 and E.coli SG21058/ptrpIL-2X-M-CSF406!.

These transformants were cultured in LB medium (T. Maniatis, E. F.Fritsch and J. Sambrook, Molecular Cloning, A Laboratory Manual, p. 440(1982), Cold Spring Harbor Laboratory) containing 50 μg/ml ampicillinovernight at 37° C. A 0.5-ml portion of each culture was added to 50 mlof M9 medium (cf. the reference cited above) with 1% casamino acid, 5μg/ml thiamine hydrochloride, 20 μg/ml L-cysteine and 50 μg/mlampicillin added and the glucose concentration adjusted to 0.4% and,after 8 hours of shake culture at 37° C., cells were recovered bycentrifugation (5,000 rpm), and the protein produced was analyzed, inthe same manner as in Reference Example 5-(7), by SDS-PAGE and Westernblotting.

As a result, the M-CSFs each encoded in the second cistron were detectedin positions corresponding to the respective expected molecular weightsfor E. coli SG21058/ptrpIL-2X-M-CSF109, E. coliSG21058/ptrpIL-2X-M-CSF402, E. coli SG21058/ptrpIL-2X-M-CSF403, E. coliSG21058/ptrpIL-2X-M-CSF404, E. coli SG21058/ptrpIL-2X-M-CSF405 and E.coli SG21058/ptrpIL-2X-M-CSF406.

(8) Isolation and purification of M-CSF derivative (E. coli4-153!-M-CSF)

1! Preparation of M-CSF fraction from E. coli

To 7.5 g (wet weight) of E. coli SG21058 strain carrying the plasmidptrpIL-2X-M-CSF109 obtained by the above procedure (1) was added 50 mMTris-hydrochloride buffer (pH 7.0) to make the whole volume 100 ml,followed by thorough stirring. Then, 6 ml of 2 mg/ml lysozyme was added,and then 4 ml of 0.14M EDTA was added. The mixture was stirred at 4° C.for 15 minutes, and then sonicated (20 KHz, 10 minutes, 200 W) andfurther centrifuged at 10,000 revolutions/minute for 20 minutes to givea pellet. This was further washed with a buffer for washing (50 mMTris-hydrochloride buffer containing 2% Triton X-100, pH 7.0) and againcentrifuged under the same conditions. After repeating this proceduretwice, an M-CSF fraction (pellet) was obtained.

2! Refolding of M-CSF from M-CSF fraction

To the M-CSF fraction obtained by 1! above was added 20 ml of 50 mMTris-hydrochloride buffer (pH 7.0) containing 7M guanidine hydrochlorideand 25 mM 2-mercaptoethanol, and the mixture was stirred at roomtemperature for at least 4 hours for reducing, denaturing and dissolvingthe protein. This solution was gradually added dropwise into a breakeralready containing 2,000 ml of 50 nM Tris-hydrochloride (pH 8.5)containing 0.5 mM reduced-form glutathione, 0.1 mM oxidized-formglutatione and 2M urea (with agitation with a stirrer) and the resultantmixture was allowed to stand at 4° C. for at least 2 days. The solutionwas then centrifuged at 3,500 revolutions/minute for 30 minutes, and thepellet was removed and the supernatant was recovered.

The refolded M-CSF was present in the thus-obtained supernatant.

3! Purification of M-CSF

The centrifugation supernatant obtained by 2! above was purifed in thefollowing manner.

3-1! Hydrophobic high-performance liquid chromatography

The above centrifugation supernatant was concentrated by anultrafiltration device (product of Amicon, with a YM-10 membrane(Amicon) used), and ammonium sulfate was added to the concentrate tomake a 30%-saturated solution. The resultant solution was centrifuged at10,000 revolutions/minute for 20 minutes. The pellet was removed and thesupernatant was recovered. This supernatant was passed through a 0.45-μmMillipore filter and then subjected, in two portions, to hydrophobicinteraction chromatography under the following conditions.

Column: TSKgel Phenyl 5PW (7.5 mm ID×75 mm, product of TosohCorporation)

Eluent A: 30% Ammonium sulfate-saturated 40 mM sodium phosphate buffer(pH 7.4)

Eluent B: 40 mM Sodium phosphate buffer (pH 7.4)

Flow rate: 1.0 ml/min.

Fraction volume: 1 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min.)                                                                             B %                                               ______________________________________                                                         0      0                                                                      7      0                                                                     40      100                                                                   45      100                                                                   50      0                                                     ______________________________________                                    

As a result, an M-CSF activity was eluted in fractions corresponding tothe ammonium sulfate concentration of 11-6%. Said active fractions werecollected, and subjected to buffer exchange for 40 mm sodium phosphate(pH 7.4) using the above-mentioned ultrafiltration device.

3-2! Anion exchange high-performance liquid chromatography

The fraction obtained by the above procedure 3-1! was subjected, in 4portions, to DEAE-5PW anion exchange high-performance liquidchromatography under the following conditions.

Column: TSKgel DEAE-5PW (7.5 mm ID×75 mm, product of Tosoh Corporation)

Eluent A: 50 mM Sodium phosphate buffer (pH 7.4)

Eluent B: 50 mM Sodium phosphate buffer (pH 7.4) containing 1.0M NaCl

Flow rate: 1.0 ml/min.

Fraction volume: 1.0 ml/tube/min.

    ______________________________________                                        Concentration gradient:                                                                         Time (min.)                                                                             B %                                               ______________________________________                                                         0      0                                                                      5      0                                                                     55      30                                                                    60      100                                                                   65      100                                                                   70      0                                                     ______________________________________                                    

From the result (elution pattern) of the above DEAE-5PW ion exchangehigh-performance liquid chromatography, the peaks observed in fractionsNos. 41 and 42 (NaCl concentration 0.21-0.22M) were found to correspondto M-CSF. Said peaks were collected to give the purified human M-CSFderivative (E. coli 4-153!-M-CSF) of the invention.

(9) Determination of N-terminal amino acid sequence of M-CSF derivative(E. coli 4-153!-M-CSF)

The N-terminal amino acid sequence of the M-CSF derivative (E. coli4-153!-M-CSF) obtained by the above procedure (8)- 3-2! was determinedusing a gaseous phase sequencer (product of Applied Biosystems).

As a result, the sequence Ser-Glu-Tyr-X'-Ser and the sequenceMet-Ser-Glu-Tyr-X'-Ser were found in a ratio of about 7-8 to 1.

In the sequences mentioned above, the amino acid X' could not beidentified but was estimated to be Cys based on the gene structure.

Bioavailability

Bioavailability serves as an index of whether or not the drugadministered is effectively utilized in the living body.

Herein, the bioavailability is expressed by the ratio of blood half-lifein the case of intravenous injection and blood half-life in the case ofsubcutaneous injection in terms of integration value.

The blood half-life was determined by RIA. The integration value of theblood half-life was determined by the trapezoidal rule, andbioavailability was calculated according to the following equation.

    Bioavailability (%)=AUC.sub.SC /AUC.sub.iv ×100

(1) Blood half-life measurement

The M-CSF derivative (E. coli 3-153!-M-CSF) and CHO -32-522!-M-CSFobtained in Reference Example 1 were each diluted with MSA (mouse serumalbumin)-containing physiological saline (30 μg/ml) to a concentrationof 20 μg/ml and these were administered to groups of 4 mice (BALB/cmales, 7 weeks of age) intravenously (i.v.) at a dose of 0.25 ml permouse (200 μg/kg). After administration, the amount of M-CSF in bloodwas determined by RIA at timed intervals.

The blood half-life measurment by said RIA was carried out by thefollowing method. Thus, a mixture of 100 μl of the test sample (serum)or a standard M-CSF (M-CSF derivative (E. coli 3-153!-M-CSF) obtained inReference Example 2) solution, 100 1 of a solution containing 10,000 cpm(per 100 μl) of Na¹²⁵ I-labeled M-CSF (derived from the above-mentionedstandard M-CSF by labeling with ¹²⁵ I by the iodogen method) and 200 μlof an anti-M-CSF rabbit antiserum (prepared by 40,000-fold dilution ofthe polyclonal antibody OCT511 obtained by the method to be mentionedlater herein with 0.1% BSA/PBS/0.05% thimerosal) is allowed to stand atroom temperature for 20 hours or at 4° C. for 48 hours to thereby allowthe antigen-antibody reaction to occur. After completion of thereaction, the labeled M-CSF-bound product and the non-bound product areseparated by the two-antibody method. For this separation, 100 μl of anormal rabbit serum 400-fold diluted with 0.1% BSA/PBS/0.05% thimerosal,100 μl of an anti-rabbit IgG serum 40-fold diluted in the same mannerand 200 μl of 12.5% polyethylene glycol solution in PBS are first added,the reaction is allowed to proceed at 4° C. for 30 minutes, andcentrifugation is performed at 3,000 rpm for 15 minutes, followed bydecantation to give a labeled M-CSF-antibody conjugate as theprecipitate. The precipitate is counted with a gamma counter for 1minute. A standard curve is prepared using standard solutions, and thenumber of counts measured similarly by repeating the above procedurewith respect to the test sample containing the M-CSF derivative of theinvention is plotted on the standard curve, and the M-CSF concentrationin said test sample is determined.

The results thus obtained are shown below in Table 6.

                  TABLE 6                                                         ______________________________________                                                 CHO              E. coli                                             Time      -32-522!-        3-153!-                                                                              E. coli 3-214!-                             (min)    M-CSF            M-CSF   M-CSF                                       ______________________________________                                               M-CSF (I.V. Administration)                                             5       1335             2078      3850                                       15      891              304       3368                                       30      623              41        2715                                       60      434              12        1751                                      120      272              4         447                                       240      44               --        124                                       360      10               --        19                                        Half-life                                                                              53.0    min      14.4 min. 46.2  min                                        M-CSF (S.C. Administration)                                             15      --               17.21     --                                         30      1.52             58.85     6.55                                       60      1.65             78.63     24.27                                     120      4.61             98.12     108.91                                    240      9.81             56.88     138.33                                    480      11.32            4.70      62.33                                     720      7.16             --        --                                        Half-life                                                                              362.9   min      79.5 min  208.7 min                                 ______________________________________                                    

In the table, the unit is ng/ml.

Bioavailabilities of CHO -32-522!-M-CSF and E. coli 3-153!-M-CSF werecalculated from the results of Table 6 above with the results shownbelow,

CHO -32-522!-M-CSF:

    (5876.6/85087.5)×100=6.63%

E. coli 3-1531!-M-CSF:

    (24519.9/20966.5)×100=116.95%

E. coli 3-214!-M-CSF:

    (44602.0/267107.0)×100=16.70%

As clear from the above, the M-CSF derivative E. coli 3-153!-M-CSF ofthe invention has a good delivery into blood by subcutaneousadministration, and is suggested to have superior bioavailability bysubcutaneous administration to that of CHO -32-522!-M-CSF, and thereforeit is believed to be particularly suitable as an anti-allergic agentwhich is usually administered subcutaneously.

The polyclonal antibody against M-CSF as used in the above RIA has thefollowing properties.

1) The polyclonal antibody used in the above procedure was prepared bythe following method.

For polyclonal antibody production, female New Zealand white rabbitsweighing 2.5 kg to 3.0 kg were subjected to multipoint intracutaneousimmunization with 20 μg/animal of a purified sample of CHO-32-522!-M-CSF (dissolved in PBS) together with the equal amount ofFreund's complete adjuvant, followed by immunizations with 20 μg of thesame sample together with Freund's imcomplete adjuvant at one-monthintervals. The number of immunizations, inclusive of the first one,amounted to 7. One week after completion of the immunization procedure,the whole blood was collected from the rabbits and antisera wereobtained.

One of the three antibodies obtained was designated as OCT511 and storedat -80° C.

2) The polyclonal antibody OCT511 to be used in the determination ofblood half-life of each of the M-CSF derivatives has the followingproperties.

1! Antibody titer

CHO -32-522!-M-CSF was labeled with ¹²⁵ I by the Iodogen method and theantibody titer was defined as the dilution factor for the antiserumcapable of binding 50% of 10 kcpm of this ¹²⁵ I-labeled CHO-32-522!-M-CSF.

As a result, the antibody titer of OCT511 was 80,000.

2! Neutralizing activity

The neutralizing activity was determined by the colony assay methodusing mouse bone marrow cells. The neutralizing activity of OCT511 wassuch that 1 ml of OCT511 could neutralize 1 to 2×10⁶ units of CHO-32-522!-M-CSF.

3! Cross-reactivity

This OCT511 did not cross-react with mouse CSF (L-cell culturesupernatant) or with human GM-CSF (Amersham) at all. Furthermore, it didnot cross-react at all with human IL-1α (cf. Unexamined Japanese PatentPublication SHO 63-164899), IL-1β (cf. Unexamined Japanese PatentPublication SHO 63-152398), IL-2 (Amersham) or TNF-α (Amersham).

EXAMPLE 5

The effect of E. coli 3-153!M-CSF on picryl chloride-induced delayedtype hypersensitive skin reaction (PC-DTH) in mouse which is widely usedas an assay for cellular immunity was investigated.

The following materials were used.

Mice: 8-week-old male BALB/c mice (SL C), 5 mice in each group, wereused.

Antigen solution: As an antigen for sensitization, a solution preparedby dissolving picryl chloride (PC) (product of Nakarai Kagaku Yakuhin)in ethanol to a concentration of 0.5% (w/v) was used. As an antigen forchallenge, a solution of picryl chloride dissolved in olive oil to aconcentration of 1% (w/v) was used.

Apparatus for measurement:

Dial thickness gauge (product of Ozaki Seisakusho) was used

Method of sensitization:

Sensitization was conducted by application to the ears of mice. Theapplication was carried out according to the method of Tajima et al(Shigeru TAJIMA, Eiko IMAI, Keizo ITO and Takashi NOSE: "Simplifiedmethod in sensitization to picryl chloride for induction of delayed typehypersensitivity in mice", Igaku no Ayumi, Vol. 122, No. 4, pp 240-242,1982).

That is to say, the antigen for sensitization was applied to the frontand rear sides of the right ear each in an amount of 10 μl withmicropipet. Four days after the sensitization, the thickness of the leftear was measured. This was used as the pre-reaction value. Thereafter,the above antigen solution for challenge was applied to the front andrear sides of the left ear each in an amount of 5 μl, and 24 hoursthereafter the thickness of the left ear was measured, and thedifference (ΔT) between this thickness and the pre-reaction value wasdesignated increase in the ear thickness.

The percent inhibition relative to the control group can be determinedby the following equation.

Percent inhibition (%)

    After Control=1-(ΔT of M-CSF-treated group/ΔT of control)×100

E. coli 3-153!M-CSF was administered in the following manner.

E. coli 3-153!M-CSF was adjusted to a concentration of 10 or 100μg/kgper mouse with physiological saline containing mouse serum albumin (MSA)and administered subcutaneously. The treated groups were as follows.

The group that received the solvent (physiological saline containing 30μg/ml MSA) was used as control group. Said group was treated in the samemanner as the following treated group No. 1.

Treated Groups

No. 1: a group in which the drug was given after sensitization andthereafter administered for 3 consecutive days with challenge conductedon the fourth day.

No. 2: a group in which the drug was given after sensitization andthereafter administered for 3 consecutive days with challenge conductedon the fourth day, followed by a single administration.

No. 3: a group in which the drug was administered for 3 consecutive daysbefore sensitization and again administered after the sensitization andfor 3 consecutive days thereafter with challenge conducted on the fourthday.

No. 4: a group in which the drug was administered for 3 consecutive daysbefore sensitization and again administered after the sensitization andfor 3 consecutive days thereafter with challenge conducted on the fourthday, followed by a single administration.

The results are shown in Table 7 below.

                  TABLE 7                                                         ______________________________________                                                            Increase in                                                                   ear thickness                                             ______________________________________                                        Administration of M-CSF (10 μg/kg)                                         Treated Group 1       12.1 ± 0.6                                           Treated Group 2       12.1 ± 1.8                                           Treated Group 3       12.9 ± 0.5                                           Treated Group 4        9.1 ± 1.4                                           Administration of M-CSF (100 μg/kg)                                        Treated group 1        8.8 ± 1.7                                           Treated group 2       13.4 ± 2.3                                           Treated group 3       14.2 ± 0.3                                           Treated group 4       10.1 ± 1.1                                           Control group         20.1 ± 1.5                                           ______________________________________                                    

The increase in ear thickness is expressed in terms of mean±SE and theunit thereof is ×10² /mm.

As a result of this example, induction of DTH was inhibited in any ofthe treated groups to a larger extent compared with the control group.

In treated groups 1 wherein M-CSF was administered at a dose of 10 or100 μg/kg, inhibitions of comparable degree were observed. In treatedgroups 2 and 3, stronger inhibition was observed at 10 μ/kg rather thanat 100 μg/kg. In treated groups 4, about 50% inhibition was observed at10 and 100 μg/kg.

The above results suggests that the induction of DTH with M-CSF isstrongly inhibited in the group in which M-CSF was administered beforesensitization and, after sensitization, consecutively administered,followed by challenge and subsequent single administration.

EXAMPLE 6

The administration timing of E. coli 3-153!M-CSF was checked withrespect to its effect on mouse PC-DTH using a method of Example 5.

E. coli 3-153!M-CSF was diluted with physiological saline containing MSA(30 μg MSA/ml) and administered at doses of 1, 10 and 100 μg/kg with theadministration timing as shown below via subcutaneous route (sc) while agroup that received a solvent (physiological saline containing MSA)served as a control group.

Treated Group

I: a group wherein the drug was administered after sensitization withchallenge conducted on the fourth day.

II: a group wherein the drug was administered after sensitization andthereafter administered for 2 consencutive days with challenge conductedon the fourth day.

III: a group wherein the drug was administered after sensitization andthereafter administered for 3 consecutive days with challenge and drugadministration conducted on the fourth day.

IV: a group which is treated in the same manner as in treated group IIIexcept that the drug was administered twice a day.

V: a group wherein the drug was not administered before challenge, butadministered three times at the time of challenge, i.e., 4 hours beforechallenge, concurrently with challenge and 4 hours after challenge.

Table 8 shows the results.

                  TABLE 8                                                         ______________________________________                                                  E. coli 3-153! M-CSF (S. C. administration)                         Group solvent   1 μg/kg 10 μg/kg                                                                            100 μg/kg                              ______________________________________                                        I     21.6 ± 2.9                                                                           24.2 ± 1.9                                                                            18.5 ± 4.8                                                                          18.4 ± 3.7                                                        (14.4%)  (14.8%)                                   II    26.6 ± 2.2                                                                           25.0 ± 3.0                                                                             18.6 ± 2.2*                                                                         13.8 ± 1.9**                                           (6.0%)    (30.1%)  (48.1%)                                   III   24.0 ± 1.1                                                                            15.3 ± 1.3**                                                                         15.5 ± 2.0                                                                           13.1 ± 2.6*                                           (36.3%)    (35.4%)  (45.4%)                                   IV    23.8 ± 1.0                                                                           21.3 ± 1.3                                                                             16.5 ± 1.5**                                                                        13.4 ± 2.8*                                           (10.5%)    (30.7%)  (43.7%)                                   V     26.7 ± 1.3                                                                           24.2 ± 2.0                                                                            23.6 ± 3.5                                                                           18.7 ± 2.5*                                            (9.4%)    (11.6%)  (30.0%)                                   ______________________________________                                         The values indicate increase in ear thickness (Mean ± SE), and the uni     is × 10.sup.2 mm.sup.2                                                  The values in parentheses show percent inhibition.                            *: p < 0.05                                                                   **: p < 0.01                                                             

As a result, significant inhibitory effect was observed at a dose of 10μg/kg or more in groups II to IV wherein the drug was administered 3times or 5 times or administered 5 times, each twice a day during the 4days period from the primary sensitization to the secondarysensitization. Furthermore, significant inhibitory effect was alsoobserved in group V wherein the drug was administered after theestablishment of DTH.

EXAMPLE 7

Three kinds of M-CSFs, i.e., E. coli 3-153!M-CSF, E. coli 3-214!M-CSFand CHO -32-522! were tested for ability to inhibit mouse PC-DTH.

Each of the M-CSFs was diluted with plysiological saline containing MSA(30 μg/ml) and administered at doses of 1, 10 and 100 μg/kgsubcutaneously (SC) and interavenously (IV).

The administration timing was the same as treated group III in Example6. Namely, the drug was administered after sensitization and for 3consecutive days thereafter, followed by challenge and administration onthe fourth day. Other procedures are the same as in Examples 5 and 6.

The results are shown in Table 9 and Table 10.

In the case of intravenous administration, CHO(-32-522)M-CSF and E. coli3-214!M-CSF exhibited dose-dependent inhibition tendency and achievedclear inhibition at 100 μg/kg. While E. coli 3-153!M-CSF exhibitedinhibition at lower doses (1 μ/g, 10 μg/kg), the degree of inhibitionwas weak.

In the case of subcutaneous administration, the 3 kinds of M-CSFsexhibited inhibition tendency, and strong ihibition was observed onlyfor E. coli 3-153!M-CSF. It became clear from the foregoing that the DTHinhibition reaction is common to the M-CSFs and that E. coli 3-153!M-CSFis excellent in the case of subcutaneous administration. This result isin agreement with the results of bioavailability obtained bysubcutaneous administration (Table 6). It is strongly suggested that E.coli 3-153!M-CSF is a derivative having excellent propertiesparticularly as M-CSF for subcutaneous injection.

                  TABLE 9                                                         ______________________________________                                        Intravenous Administration                                                            1 μg/kg                                                                             10 μg/kg 100 μg/kg                                     ______________________________________                                        CHO(-32-522)                                                                  M-CSF     28.4 ± 1.4                                                                            25.3 ± 1.2                                                                             21.8 ± 1.8*                               (Lot 9J-72)          (10.0%)     (22.4%)                                      E. coli(3-214)                                                                M-CSF     26.3 ± 1.5                                                                            24.3 ± 1.0                                                                              18.7 ± 1.8**                             (Lot 9I-98)                                                                             (6.4%)     (13.5%)     (33.5%)                                      E. coli(3-153)                                                                M-CSF      24.2 ± 0.6*                                                                           21.0 ± 1.7*                                                                           23.7 ± 2.6                                (Lot 9J-81)                                                                             (13.9%)    (25.3%)     (15.7%)                                      Solvent              28.1 ± 1.5                                            ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Subcutaneous Administration                                                            1 μg/kg                                                                              10 μg/kg                                                                             100 μg/kg                                     ______________________________________                                        CHO(-32-522)                                                                  M-CSF      27.2 ± 0.7                                                                             22.5 ± 4.1                                                                           24.8 ± 1.9*                               (Lot 9J-72)                                                                              (0%)        (17.3%)   (8.8%)                                       E. coli(3-214)                                                                M-CSF      22.7 ± 1.7                                                                             23.1 ± 1.5                                                                           22.5 ± 1.4                                (Lot 9I-98)                                                                              (16.5%)     (15.1%)   (17.3%)                                      E. coli(3-153)                                                                M-CSF       22.6 ± 2.5*                                                                            23.5 ± 1.5*                                                                         17.3 ± 2.1*                               (Lot 9J-81)                                                                              (16.9%)     (13.6%)   (36.4%)                                      Solvent                27.2 ± 1.8                                          ______________________________________                                    

The values indicate increase in ear thickness (Mean ±SE). The unit is×10⁻² mm². The values in parentheses shore percent inhibition.

* p<0.05,

** p<0.01

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 554 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetThrAlaProGlyAlaAlaGlyArgCysProProThrThrTrpLeu                              151015                                                                        GlySerLeuLeuLeuLeuValCysLeuLeuAlaSerArgSerIleThr                              202530                                                                        GluGluValSerGluTyrCysSerHisMetIleGlySerGlyHisLeu                              354045                                                                        GlnSerLeuGlnArgLeuIleAspSerGlnMetGluThrSerCysGln                              505560                                                                        IleThrPheGluPheValAspGlnGluGlnLeuLysAspProValCys                              65707580                                                                      TyrLeuLysLysAlaPheLeuLeuValGlnXaaIleMetGluAspThr                              859095                                                                        MetArgPheArgAspAsnThrProAsnAlaIleAlaIleValGlnLeu                              100105110                                                                     GlnGluLeuSerLeuArgLeuLysSerCysPheThrLysAspTyrGlu                              115120125                                                                     GluHisAspLysAlaCysValArgThrPheTyrGluThrProLeuGln                              130135140                                                                     LeuLeuGluLysValLysAsnValPheAsnGluThrLysAsnLeuLeu                              145150155160                                                                  AspLysAspTrpAsnIlePheSerLysAsnCysAsnAsnSerPheAla                              165170175                                                                     GluCysSerSerGlnAspValValThrLysProAspCysAsnCysLeu                              180185190                                                                     TyrProLysAlaIleProSerSerAspProAlaSerValSerProHis                              195200205                                                                     GlnProLeuAlaProSerMetAlaProValAlaGlyLeuThrTrpGlu                              210215220                                                                     AspSerGluGlyThrGluGlySerSerLeuLeuProGlyGluGlnPro                              225230235240                                                                  LeuHisThrValAspProGlySerAlaLysGlnArgProProArgSer                              245250255                                                                     ThrCysGlnSerPheGluProProGluThrProValValLysAspSer                              260265270                                                                     ThrIleGlyGlySerProGlnProArgProSerValGlyAlaPheAsn                              275280285                                                                     ProGlyMetGluAspIleLeuAspSerAlaMetGlyThrAsnTrpVal                              290295300                                                                     ProGluGluAlaSerGlyGluAlaSerGluIleProValProGlnGly                              305310315320                                                                  ThrGluLeuSerProSerArgProGlyGlyGlySerMetGlnThrGlu                              325330335                                                                     ProAlaArgProSerAsnPheLeuSerAlaSerSerProLeuProAla                              340345350                                                                     SerAlaLysGlyGlnGlnProAlaAspValThrGlyThrAlaLeuPro                              355360365                                                                     ArgValGlyProValArgProThrGlyGlnAspTrpAsnHisThrPro                              370375380                                                                     GlnLysThrAspHisProSerAlaLeuLeuArgAspProProGluPro                              385390395400                                                                  GlySerProArgIleSerSerProArgProGlnGlyLeuSerAsnPro                              405410415                                                                     SerThrLeuSerAlaGlnProGlnLeuSerArgSerHisSerSerGly                              420425430                                                                     SerValLeuProLeuGlyGluLeuGluGlyArgArgSerThrArgAsp                              435440445                                                                     ArgArgSerProAlaGluProGluGlyGlyProAlaSerGluGlyAla                              450455460                                                                     AlaArgProLeuProArgPheAsnSerValProLeuThrAspThrGly                              465470475480                                                                  HisGluArgGlnSerGluGlySerSerSerProGlnLeuGlnGluSer                              485490495                                                                     ValPheHisLeuLeuValProSerValIleLeuValLeuLeuAlaVal                              500505510                                                                     GlyGlyLeuLeuPheTyrArgTrpArgArgArgSerHisGlnGluPro                              515520525                                                                     GlnArgAlaAspSerProLeuGluGlnProGluGlySerProLeuThr                              530535540                                                                     GlnAspAspArgGlnValGluLeuProVal                                                545550                                                                        __________________________________________________________________________

We claim:
 1. A method of treating a Type I allergy or a Type IV allergycomprising administering biologically active macrophage-colonystimulating factor (M-CSF), wherein said macrophage-colony stimulatingfactor is a macrophage-colony stimulating factor having an amino acidprimary sequence of Val at position 3 or Ser at position 4 to Thr atposition 153 or an amino acid primary sequence of Val at position 3 orSer at position 4 to Pro at position 214, or an amino acid primarysequence of Met at position -32 to Val at position 522 of the formula(1), wherein formula (1) is: ##STR4## wherein x is Tyr or Asp.
 2. Themethod of claim 1 wherein the macrophage-colony stimulating factor is amacrophage-colony stimulating factor having an amino acid primarysequence of Val at position 3 or Ser at position 4 to Thr at position153 or an amino acid primary sequence of Val at position 3 or Ser atposition 4 to Pro at position 214 of the formula (1).
 3. The method ofclaim 1 wherein the macrophage-colony stimulating factor is amacrophage-colony stimulating factor having an amino acid primarysequence of Val at position 3 or Ser at position 4 to Thr at position153 of the formula (1).
 4. The method of claim 1 wherein themacrophage-colony stimulating factor is a macrophage-colony stimulatingfactor having an amino acid primary sequence of Val at position 3 or Serat position 4 to Pro at position 214 of the formula (1).
 5. The methodof claim 1 wherein the macrophage-colony stimulating factor is amacrophage-colony stimulating factor having an amino acid primarysequence of Val at position 3 to Thr at position 153 of the formula (1).6. The method of claim 1, wherein the allergy is Type I allergy.
 7. Themethod of claim 1, wherein the allergy is Type IV allergy.
 8. The methodof claim 1, wherein the macrophage-colony stimulating factor is amacrophage-colony stimulating factor having a amino acid primarysequence of Met at position -32 to Val at position 522 of the formula(1).